CD37-binding molecules and immunoconjugates thereof

ABSTRACT

Novel anti-cancer agents, including, but not limited to, antibodies and immunoconjugates, that bind to CD37 are provided. Methods of using the agents, antibodies, or immunoconjugates, such as methods of inhibiting tumor growth are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 61/313,628, filed Mar. 12, 2010, U.S. ProvisionalApplication No. 61/327,314, filed Apr. 23, 2010, and U.S. ProvisionalApplication No. 61/412,644, filed Nov. 11, 2010, each of which is herebyincorporated by reference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted substitute sequence listing(Name: 2921.0030003Revisedsequencelisting.ascii.txt, Size 251,449 bytes;and Date of Creation: Jun. 12, 2013) is herein incorporated by referencein its entirety.

FIELD OF THE INVENTION

The field of the invention generally relates to antibodies,antigen-binding fragments thereof, polypeptides, and immunoconjugatesthat bind to CD37, as well as to methods of using such CD37-bindingmolecules for the treatment of diseases, such as B-cell malignancies.

BACKGROUND OF THE INVENTION

Leukocyte antigen CD37 (“CD37”), also known as GP52-40, tetraspanin-26,or TSPAN26, is a transmembrane protein of the tetraspanin superfamily(Maecker et al., 1997 FASEB J. 11:428-442). It is a heavily glycosylatedprotein with four transmembrane domains that is expressed on B cellsduring the pre-B to peripheral mature B-cell stages, but is absent onterminal differentiation to plasma cells. (Link et al., 1987, J Pathol.152:12-21). The CD37 antigen is only weakly expressed on T-cells,myeloid cells and granulocytes (Schwartz-Albiez et al. 1988, J.Immunol., 140(3)905-914). However, CD37 is also expressed on malignantB-cells such as those founding non-Hodgkin's lymphoma (NHL) and chroniclymphoid leukemia (CLL) (Moore et al. 1986, J Immunol. 137(9):3013-8).This expression profile suggests that CD37 represents a promisingtherapeutic target for B-cell malignancies.

While the exact physiological role of CD37 in unclear, studies suggest apotential role in T-cell proliferation (van Spriel et al. 2004, JImmunol., 172(5):2953-61) As part of the tetraspanin family of cellsurface glycoproteins, CD37 may also complex with other surface proteins(Angelisová 1994, Immunogenetics., 39(4):249-56). Mice deficient in CD37expression were developed and revealed no changes in development andcellular composition of lymphoid organs. Only reduced levels of IgG1 andalterations of responses to T-cell dependent antigens were observed(Knobeloch et al. 2000, Mol Cell Biol., 20(15):5363-9).

Antibodies are emerging as a promising method to treat such cancers. Inparticular, antibodies that are able to induce apoptosis in target cellsare desirable. In addition, antibodies having complement-dependentcytotoxicity (CDC) activity and antibody-dependent cytotoxicity (ADCC)are also desirable.

Currently, an anti-CD20 antibody called rituximab is being used to treatB-cell malignancies (Leget et al., 1998, Curr. Opin. Oncol.,10:548-551). However, only a subset of patients respond to rituximabtreatment, and even responding patients taking rituximab eventuallyrelapse and often develop resistance to rituximab treatment. Inaddition, CD37-binding agents are also being tested as potentialtherapeutics for B-cell malignancies. Trubion Pharmaceuticals developedthe CD37-binding agents SMIP-016 and TRU-016 (Zhao et al., 2007, Blood,110:2569-2577). SMIP-016 is a single chain polypeptide that includesvariable regions from a hybridoma and engineered human constant regions.TRU-016 is a humanized version of the anti-CD37 SMIP protein. See e.g.U.S. Published Application No. 2007/0009519. TRU-016 is being testedclinically for the treatment of chronic lymphocytic leukemia (CLL).Boehringer Ingelheim has also disclosed a CD37 binding agent inInternational Published Application No. WO 2009/019312. However, no CDCactivity has been described for any of these binding agents and no invitro pro-apoptotic activity has been described in the absence ofcross-linking agents.

Radio-immunotherapy (RIT) has been attempted using a radio-labeledanti-CD37 antibody MB-1 in two separate trials. Therapeutic doses of¹³¹I-MB-1 were administered to six relapsed NHL patients (Press et al.1989 J Clin Oncol. 7(8):1027-38, Press at el. 1993, N Engl J Med.329(17):1219-24). All six patients achieved a complete remission (CR)with a duration of four to thirty-one months. In another trial,¹³¹I-MB-1 was administered to ten relapsed NHL patients (Kaminski et al.1992 J Clin Oncol. 10(11):1696-711). A total of four patients had aresponse ranging in duration from two to six months, although only oneCR was reported. However, not all patients could be treated due to anunfavorable biodistribution of the radio-label which raised concern forradiation exposure of vital non-target organs. Indeed, RIT relatedtoxicities were observed in these trials including severemyelosupression and cardiopulmonary toxicity. While these clinical datasuggest that anti-CD37 radio-immunoconjugates may be effective, thesetherapies are cumbersome to administer, and at relapse post-RIT patientscannot be retreated with RIT due to the risks associated with high dosesof radiation.

To overcome the limitations of RIT, antibody-cytotoxic agent conjugates(ACC), also called antibody-drug conjugates (ADC), have been developed.These are immunoconjugates that include a cytotoxic agent covalentlylinked to an antibody through a chemical linker which can allow forspecific delivery of cytotoxic drugs to cells expressing a proteinrecognized by the antibody. However, proteins that are poorlyinternalized are not considered to be favorable targets for suchtherapeutics. CD37 is structurally similar to CD20 as both antigenscontain four transmembrane domains, although CD20 is not part of thetetraspanin family (Tedder et al. 1989, J. Immun. 142: 2560-2568).Antibodies against several B-cell antigens including CD37 and CD20 havebeen studied for their ability to undergo endocytosis and degradation(Press et al. 1989, Cancer Res. 49(17):4906-12, and Press et al. 1994,Blood. 83(5):1390-7). The anti-CD37 antibody MB-1 was retained on thecell surface and internalized slowly in Daudi lymphoma cells in vitro.The MB-1 antibody also had a low rate of endocytosis and intracellularmetabolism in NHL patient cells in vitro. Similar results were obtainedwith the anti-CD20 antibody 1F5, which was also retained mainly on thelymphoma cell surface and internalized poorly. ADCs of CD20 antibodieshave been studied previously but have not demonstrated significantlystrong potency, especially when non-disulfide or acid stable linkers areused (see for example Polson et al., 2009, Cancer Res.,69(6):2358-2364). In light of these observations, CD37 has not beenconsidered a favorable target for antibody-drug conjugates.

Therefore, there exists a need for CD37 binding agents includingantibodies, antigen-binding fragments thereof, and antibody-drugconjugates (immunoconjugates) as a means to treat B-cell malignancies.The present invention addresses that need.

BRIEF SUMMARY OF THE INVENTION

Novel antibodies that bind to human CD37, immunoconjugates comprisingthese antibodies, and methods of their use are described herein. Novelpolypeptides, such as antibodies that bind human CD37, fragments of suchantibodies, and other polypeptides related to such antibodies are alsoprovided. Polynucleotides comprising nucleic acid sequences encoding thepolypeptides are also provided, as are vectors comprising thepolynucleotides. Cells comprising the polypeptides and/orpolynucleotides of the invention are further provided. Compositions(e.g., pharmaceutical compositions) comprising the novel CD37 antibodiesor immunoconjugates are also provided. In addition, methods of makingand using the novel CD37 antibodies or immunoconjugates are alsoprovided, such as methods of using the novel CD37 antibodies orimmunoconjugates to inhibit tumor growth and/or treat cancer.

Antibodies or antigen binding fragment thereof that specifically bind toCD37, and are capable of inducing complement dependent cytotoxicity(CDC) are provided. In some embodiments, the antibody is also capable ofinducing apoptosis and/or antibody dependent cell mediated cytotoxicity(ADCC).

The antibody or antigen binding fragment thereof can be one thatspecifically binds to the same CD37 epitope as an antibody selected fromthe group consisting of: (a) an antibody comprising the polypeptide ofSEQ ID NO:55 and the polypeptide of SEQ ID NO:72; (b) an antibodycomprising the polypeptide of SEQ ID NO:59 and the polypeptide of SEQ IDNO:75; (c) an antibody comprising the polypeptide of SEQ ID NO:61 andthe polypeptide of SEQ ID NO:77; (d) an antibody comprising thepolypeptide of SEQ ID NO:64 and the polypeptide of SEQ ID NO:80; (e) anantibody comprising the polypeptide of SEQ ID NO:66 and the polypeptideof SEQ ID NO:82; (f) an antibody comprising the polypeptide of SEQ IDNO:68 and the polypeptide of SEQ ID NO:84; and (g) an antibodycomprising the polypeptide of SEQ ID NO:70 and the polypeptide of SEQ IDNO:86.

In some embodiments, the antibody or antigen binding fragment thereofspecifically binds to CD37 and specifically binds to the polypeptide ofSEQ ID NO: 180. In a certain embodiment, the antibody or antigen bindingfragment thereof does not bind to the polypeptide of SEQ ID NO: 184.

In some embodiments, the antibody or antigen binding fragment thereofspecifically binds to CD37, and the antibody or fragment thereofcompetitively inhibits an antibody selected from the group consistingof: (a) an antibody comprising the polypeptide of SEQ ID NO:55 and thepolypeptide of SEQ ID NO:72; (b) an antibody comprising the polypeptideof SEQ ID NO:59 and the polypeptide of SEQ ID NO:75; (c) an antibodycomprising the polypeptide of SEQ ID NO:61 and the polypeptide of SEQ IDNO:77; (d) an antibody comprising the polypeptide of SEQ ID NO:64 andthe polypeptide of SEQ ID NO:80; (e) an antibody comprising thepolypeptide of SEQ ID NO:66 and the polypeptide of SEQ ID NO:82; (f) anantibody comprising the polypeptide of SEQ ID NO:68 and the polypeptideof SEQ ID NO:84; and (g) an antibody comprising the polypeptide of SEQID NO:70 and the polypeptide of SEQ ID NO:86.

In certain embodiments, the antibody or antigen binding fragment thereofis produced by hybridoma selected from the group consisting of ATCCDeposit Designation PTA-10664, deposited with the ATCC on Feb. 18, 2010,ATCC Deposit Designation PTA-10665, deposited with the ATCC on Feb. 18,2010, ATCC Deposit Designation PTA-10666, deposited with the ATCC onFeb. 18, 2010, ATCC Deposit Designation PTA-10667 deposited with theATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10668, depositedwith the ATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10669,deposited with the ATCC on Feb. 18, 2010, and ATCC Deposit DesignationPTA-10670, deposited with the ATCC on Feb. 18, 2010.

The ATCC is located at 10801 University Boulevard Manassas Va. 20110.

In some embodiments, the antibody or antigen binding fragment thereofspecifically binds to CD37, and the antibody comprises polypeptidesequences selected from the group consisting of: (a) SEQ ID NOs: 4, 5,and 6 and SEQ ID NOs: 28, 29, and 30; (b) SEQ ID NOs: 7, 8, and 9 andSEQ ID NOs: 31, 32, and 33; (c) SEQ ID NOs: 10, 11, and 12 and SEQ IDNOs: 34, 35, and 36; (d) SEQ ID NOs: 13, 14, and 15 and SEQ ID NOs: 37,38, and 39; (e) SEQ ID NOs: 13, 14, and 15 and SEQ ID NOs: 37, 40, and39; (f) SEQ ID NOs: 16, 17, and 18 and SEQ ID NOs: 41, 42, and 43; (g)SEQ ID NOs: 19, 20, and 21 and SEQ ID NOs: 44, 45, and 46; (h) SEQ IDNOs: 19, 20, and 21 and SEQ ID NOs: 44, 47, and 46; (i) SEQ ID NOs: 22,23, and 24 and SEQ ID NOs: 48, 49, and 50; (j) SEQ ID NOs: 22, 23, and24 and SEQ ID NOs: 48, 51, and 50; (k) SEQ ID NOs: 25, 26, and 27 andSEQ ID NOs: 52, 53, and 54; and (l) variants of (a) to (k) comprising 1,2, 3, or 4 conservative amino acid substitutions.

In further embodiments, the antibody or antigen binding fragment thereofcomprises polypeptide sequences that are at least 90% identical, atleast 95% identical, at least 99% identical, or identical to polypeptidesequences selected from the group consisting of: (a) SEQ ID NO:55 andSEQ ID NO:72; (b) SEQ ID NO:56 and SEQ ID NO:73; (c) SEQ ID NO:57 andSEQ ID NO:74; (d) SEQ ID NO:58 and SEQ ID NO:74; (e) SEQ ID NO:59 andSEQ ID NO:75; (f) SEQ ID NO:60 and SEQ ID NO:76; (g) SEQ ID NO:61 andSEQ ID NO:77; (h) SEQ ID NO:62 and SEQ ID NO:78; (i) SEQ ID NO:63 andSEQ ID NO:79; (j) SEQ ID NO:64 and SEQ ID NO:80; (k) SEQ ID NO:65 andSEQ ID NO:81; (l) SEQ ID NO:66 and SEQ ID NO:82; (m) SEQ ID NO:67 andSEQ ID NO:83; (n) SEQ ID NO:68 and SEQ ID NO:84; (o) SEQ ID NO:69 andSEQ ID NO:85; (p) SEQ ID NO:70 and SEQ ID NO:86; and (q) SEQ ID NO:71and SEQ ID NO:87.

In some embodiments, the antibody or antigen binding fragment thereof ismurine, non-human, humanized, chimeric, resurfaced, or human.

In some embodiments, the antibody or antibody fragment is capable ofinducing apoptosis of a cell expressing CD37 in vitro in the absence ofcross-linking agents. In some embodiments, the antibody or antigenbinding fragment is capable of inducing complement dependentcytotoxicity (CDC). In still further embodiments, the antibody orantigen binding fragment is capable of inducing antibody dependent cellmediated cytotoxicity (ADCC).

In other embodiments, the antibody or antigen binding fragment thereofis human or humanized, specifically binds to CD37, and is capable ofinducing apoptosis of a cell expressing CD37 in vitro in the absence ofcross-linking agents. In further embodiments, the human or humanizedantibody or antigen binding fragment thereof is also capable of inducingcomplement dependent cytotoxicity (CDC) and/or capable of inducingantibody dependent cell mediated cytotoxicity (ADCC).

In still other embodiments, the antibody or antigen binding fragmentthereof binds to human CD37 and macaque CD37.

In some embodiments, the antibody or antigen binding fragment thereof isa full length antibody or an antigen binding fragment. The antibody orantigen binding fragment thereof can comprise a Fab, Fab′, F(ab′)2, Fd,single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar,intrabody, IgGΔCH2, minibody, F(ab′)3, tetrabody, triabody, diabody,single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

In other embodiments, the CD37-binding agent is a polypeptide thatspecifically binds CD37, and the polypeptide comprises sequencesselected from the group consisting of: (a) SEQ ID NOs: 4, 5, and 6 andSEQ ID NOs: 28, 29, and 30; (b) SEQ ID NOs: 7, 8, and 9 and SEQ ID NOs:31, 32, and 33; (c) SEQ ID NOs: 10, 11, and 12 and SEQ ID NOs: 34, 35,and 36; (d) SEQ ID NOs: 13, 14, and 15 and SEQ ID NOs: 37, 38, and 39;(e) SEQ ID NOs: 13, 14, and 15 and SEQ ID NOs: 37, 40, and 39; (f) SEQID NOs: 16, 17, and 18 and SEQ ID NOs: 41, 42, and 43; (g) SEQ ID NOs:19, 20, and 21 and SEQ ID NOs: 44, 45, and 46; (h) SEQ ID NOs: 19, 20,and 21 and SEQ ID NOs: 44, 47, and 46; (i) SEQ ID NOs: 22, 23, and 24and SEQ ID NOs: 48, 49, and 50; (j) SEQ ID NOs: 22, 23, and 24 and SEQID NOs: 48, 51, and 50; (k) SEQ ID NOs: 25, 26, and 27 and SEQ ID NOs:52, 53, and 54; and (l) variants of (a) to (k) comprising 1, 2, 3, or 4conservative amino acid substitutions.

In other embodiments, the CD37-binding agent is a polypeptide thatspecifically binds CD37, and the polypeptide comprises sequences thatare at least 90% identical, at least 95% identical, at least 99%identical, or identical to sequences selected from the group consistingof: (a) SEQ ID NO:55 and SEQ ID NO:72; (b) SEQ ID NO:56 and SEQ IDNO:73; (c) SEQ ID NO:57 and SEQ ID NO:74; (d) SEQ ID NO:58 and SEQ IDNO:74; (e) SEQ ID NO:59 and SEQ ID NO:75; (f) SEQ ID NO:60 and SEQ IDNO:76; (g) SEQ ID NO:61 and SEQ ID NO:77; (h) SEQ ID NO:62 and SEQ IDNO:78; (i) SEQ ID NO:63 and SEQ ID NO:79; (j) SEQ ID NO:64 and SEQ IDNO:80; (k) SEQ ID NO:65 and SEQ ID NO:81; (l) SEQ ID NO:66 and SEQ IDNO:82; (m) SEQ ID NO:67 and SEQ ID NO:83; (n) SEQ ID NO:68 and SEQ IDNO:84; (o) SEQ ID NO:69 and SEQ ID NO:85; (p) SEQ ID NO:70 and SEQ IDNO:86; and (q) SEQ ID NO:71 and SEQ ID NO:87.

Cells producing the antibody or antigen binding fragment thereof or thepolypeptide can also be made and used according to the methods describedherein. The methods provide methods of making an antibody orantigen-binding fragment thereof or a polypeptide comprising (a)culturing a cell producing such a CD37-binding agent; and (b) isolatingthe antibody, antigen-binding fragment thereof, or polypeptide from thecultured cell.

In some embodiments, the CD37-binding agent is an immunoconjugate havingthe formula (A)-(L)-(C), wherein: (A) is a CD37-binding agent; (L) is alinker; and (C) is a cytotoxic agent; and wherein the linker (L) links(A) to (C).

In some embodiments, the CD37-binding agent is an immunoconjugate havingthe formula (A)-(L)-(C), wherein: (A) is an antibody or antigen bindingfragment that specifically binds to CD37; (L) is a non-cleavable linker;and (C) is a cytotoxic agent; and wherein the linker (L) links (A) to(C).

In some embodiments, the CD37-binding agent is an immunoconjugate havingthe formula (A)-(L)-(C), wherein: (A) is an antibody or antigen bindingfragment that specifically binds to CD37; (L) is a linker; and (C) is amaytansinoid; and wherein the linker (L) links (A) to (C).

The immunoconjugate linker can be a non-cleavable linker. The linker canbe selected from a group consisting of a cleavable linker, anon-cleavable linker, a hydrophilic linker, and a dicarboxylic acidbased linker The linker can be selected from the group consisting of:N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP); N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (sulfoSMCC);N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); andN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide). The linker can beN-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester(NHS-PEG4-maleimide).

The cytotoxic agent can be selected from the group consisting of amaytansinoid, maytansinoid analog, doxorubicin, a modified doxorubicin,benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin,duocarmycin analog, calicheamicin, dolastatin, dolastatin analog,aristatin, tomaymycin derivative, and leptomycin derivative or a prodrugof the agent. The cytotoxic agent can be a maytansinoid. The cytotoxicagent can be N(2)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine(DM1) or N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine(DM4).

Also provided herein is a pharmaceutical composition comprising aCD37-binding agent and a pharmaceutically acceptable carrier. Thepharmaceutical composition can comprise a second anti-cancer agent.

A diagnostic reagent comprising a CD37-binding agent which is labeled isalso provided herein. The label can be selected from the groupconsisting of a radiolabel, a fluorophore, a chromophore, an imagingagent and a metal ion.

Also provided herein is a kit comprising a CD37-binding agent.

The methods described herein include methods for inhibiting the growthof a cell expressing CD37 comprising contacting the cell with a CD37binding agent or pharmaceutical composition comprising the same.

The methods also provide methods for treating a patient having cancercomprising administering to the patient a therapeutically effectiveamount of a CD37 binding agent or pharmaceutical composition comprisingthe same to the subject.

The methods can comprise administering a second anti-cancer agent to thesubject. The second anti-cancer agent can be a chemotherapeutic agent.

The cancer can be a cancer selected from the group consisting of B celllymphomas, NHL, precursor B cell lymphoblastic leukemia/lymphoma andmature B cell neoplasms, B cell chronic lymphocytic leukemia (CLL)/smalllymphocytic lymphoma (SLL), B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicularlymphoma (FL), low grade, intermediate-grade and high-grade (FL),cutaneous follicle center lymphoma, marginal zone B cell lymphoma, MALTtype marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma,splenic type marginal zone B cell lymphoma, hairy cell leukemia, diffuselarge B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cellmyeloma, post-transplant lymphoproliferative disorder, Waldenstrom'smacroglobulinemia, and anaplastic large-cell lymphoma (ALCL).

Isolated polynucleotides comprising a sequence that encodes apolypeptide at least 90% identical, at least 95% identical, at least 99%identical, or identical to a sequence selected from the group consistingof SEQ ID NOs: 55-87 are also provided herein. The polynucleotide cancomprise a sequence that is at least 90%, at least 95% identical, atleast 99% identical, or identical to SEQ ID NOs: 121-151.

Vectors and host cells comprising such polynucleotides and vectors arealso provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 depicts the histograms for antibody binding to non-transfected300-19 control cells (left panels) and CD37-expressing 300-19 cells(right panels). Histograms are shown for staining with 10 nM ofmuCD37-3, muCD37-12, muCD37-38 and the absence of primary antibody.

FIG. 2 depicts the histograms for antibody binding to non-transfected300-19 control cells (left panels) and CD37-expressing 300-19 cells(right panels). Histograms are shown for staining with 10 nM muCD37-50,muCD37-51, muCD37-56 and muCD37-57.

FIG. 3 depicts the binding of (A) muCD37-3 and muCD37-12 and (B)muCD37-8, muCD37-10 and muCD37-14 to WSU-DLCL-2 cells as assayed by flowcytometry. Mean fluorescence intensity (MFI) is plotted for eachantibody concentration used. The binding curves were used to determinethe EC50 of antibody binding, which corresponds to the apparent Kd ofeach antibody.

FIG. 4 depicts results from an Annexin-V assay to measure induction ofapoptosis using Ramos lymphoma cells incubated with a 10 nMconcentration of (A) rituximab, muCD37-3, muCD37-8, muCD37-10, muCD37-12or muCD37-14 and (B) rituximab, huCD37-3, muCD37-38, muCD37-50,muCD37-51, muCD37-56 or muCD37-57. Control samples of untreated cells inthe absence of antibody (no Ab) are used in comparison.

FIG. 5 depicts the results from WST-8 proliferation assays on SU-DHL-4lymphoma cells incubated with varying concentrations of muCD37-3,muCD37-38, muCD37-50, muCD37-51 and muCD37-16 antibodies for 5 days.

FIG. 6 depicts a list of CD37-3 surface residues and substitutions inresurfaced versions for (A) CD37-3 VL and (B) CD37-3 VH.

FIG. 7 depicts a list of CD37-50 surface residues and substitutions inthe resurfaced version for (A) CD37-50 VL and (B) CD37-50 VH.

FIG. 8 depicts alignments of resurfaced sequences for the CD37-3 andCD37-50 variable region with their murine counterparts: A) CD37-3 lightchain variable domain; B) CD37-3 heavy chain variable domain. C) CD37-50light chain variable domain; D) CD37-50 heavy chain variable domain.Dashes “-” denote identity with the murine sequence.

FIG. 9 depicts (A) direct binding assays of muCD37-3, chCD37-3,muCD37-12 and chCD37-12 to Ramos cells as assayed by flow cytometry and(B) competitive binding assays with muCD37-3, chCD37-3, huCD37-3v1.0 andhuCD37-3v1.01 to BJAB cells in the presence of 2 nM concentration ofmuCD37-3-PE conjugates.

FIG. 10 depicts binding of anti-CD37 antibodies to 300-19 cellsexpressing the macaque CD37 antigen as assayed by flow cytometry: (A)binding of muCD37-3, muCD37-12, muCD37-38, muCD37-50, muCD37-51,muCD37-56, muCD37-57, WR17 and TRU-016 and (B) binding of huCD37-3,huCD37-38, huCD37-50, huCD37-51, huCD37-56 and huCD37-57. The bindingcurves were used to determine the EC50 of antibody binding, whichcorresponds to the apparent Kd of each antibody.

FIG. 11 depicts the results from an Annexin-V assay to measure inductionof apoptosis on Ramos lymphoma cells incubated with varyingconcentration of (A) huCD37-3, huCD37-38, huCD37-50 and (B) huCD37-51,huCD37-56, huCD36-57 and rituximab. Control samples of cells treatedwith a human IgG1 isotype control antibody (huIgG control) are used incomparison.

FIG. 12 depicts the results from WST-8 proliferation assays on (A)SU-DHL-4 and (B) DOHH-2 lymphoma cells incubated with varyingconcentrations of muCD37-3, chCD37-3, huCD37-3v1.0 and huCD37-3v1.01antibodies for 5 days.

FIG. 13 depicts the results from WST-8 proliferation assays on (A)Granta-519 and (B) SU-DHL-4 lymphoma cells incubated with varyingconcentrations of huCD37-3, TRU-016 or rituximab antibodies for 5 days.

FIG. 14 depicts the results from CDC assays on Ramos lymphoma cellsincubated with (A) huCD37-3, huCD37-38, chCD37-12 or a huIgG1 isotypecontrol antibody and (B) huCD37-38, huCD37-50, huCD37-51, huCD37-56,huCD37-57, chCD37-12 or a huIgG1 isotype control antibody in thepresence of 5% human serum as a source of complement.

FIG. 15 depicts the results from an ADCC assay on Daudi lymphoma cellsincubated with (A) huCD37-3, huCD37-38, huCD37-50, TRU-016 and (B)huCD37-51, huCD37-56, huCD37-57, TRU-016 or a human IgG1 isotype controlantibody in the presence of purified human NK cells as effector cells.

FIG. 16 depicts the alignment of the full length murine, human, andmacaca CD37 amino acid sequences. Dashes “-” denote identity with themurine sequence. The small and large extracellular domains are markedwith underlines.

FIG. 17 depicts the alignment of the large extracellular domain ofhuman, recombinant and wild type murine, macaca and the chimeric CD37sequences. Dashes “-” denote identity with the human sequence. Thepositions of the engineered restriction sites are given and the affectedresidues are underlined.

FIG. 18 depicts binding of a panel of CD37 antibodies to cellstransfected with (A) human CD37 wildtype and (B) hCD37-M3 variant asassayed by flow cytometry using 1.5 μg/mL of each antibody.

FIG. 19 depicts binding of a panel of CD37 antibodies to cellstransfected with (A) the hCD37-M1 variant and (B) the hCD37-M45 variantas assayed by flow cytometry using 1.5 μg/mL of each antibody.

FIG. 20 depicts binding of (A) huCD37-3 in comparison withhuCD37-3-SMCC-DM1 huCD37-3-SPP-DM1 and huCD37-3-sulfo-mal-DM4 and (B)huCD37-38 in comparison with huCD37-38-SMCC-DM1 to BJAB cells as assayedby flow cytometry. The binding curves were used to determine the EC50 ofantibody or conjugate binding, which corresponds to the apparent Kd ofeach.

FIG. 21 depicts the results of (A) an Annexin-V assay to measureinduction of apoptosis and (B) the results from a CDC assay. Assays wereperformed on Ramos lymphoma cells incubated with varying concentrationsof the huCD37-3, huCD37-3-SMCC-DM1, huIgG1 control antibody,huIgG1-SMCC-DM1 control conjugate, or rituximab. CDC assays wereperformed in the presence of 5% human serum as a source of complement.

FIG. 22 depicts the results from ADCC assays on (A) Daudi lymphoma cellsincubated with huCD37-3, huCD37-3-SMCC-DM1, huCD37-3-PEG4-mal-DM1,TRU-016 or a huIgG1 isotype control antibody and (B) Ramos lymphomacells incubated with huCD37-3, huCD37-3-SMCC-DM1, huCD37-3-PEG4-mal-DM1or a huIgG1 isotype control antibody in the presence of purified humanNK cells as effector cells.

FIG. 23 depicts the results from a cell cycle analysis using propridiumiodide staining on (A) BJAB cells and (B) RL cells incubated withhuCD37-3, huCD37-3-SMCC-DM1, or a non-binding huIgG1-SMCC-DM1 controlconjugate at a 10 nM concentration for 20 hours.

FIG. 24 depicts the results from a WST-8 cytotoxicity assay on (A) Daudicells incubated with huCD37-3-SMCC-DM1, huCD37-38-SMCC-DM1,huCD37-50-SMCC-DM1, huCD37-51-SMCC-DM1, huCD37-56-SMCC-DM1,huCD37-57-SMCC-DM1, and (B) Granta-519 cells incubated withhuCD37-3-SMCC-DM1, huCD37-38-SMCC-DM1, huCD37-50-SMCC-DM1,huCD37-51-SMCC-DM1, or a non-binding huIgG1-SMCC-DM1 control conjugateat concentrations ranging from 3×10⁻⁸ M to 1×10⁻¹¹ M for 5 days.

FIG. 25 depicts the results of an established xenograft model using BJABlymphoma cells implanted subcutaneous into SCID mice. Animals weretreated once on day 12 post cell inoculation with either 10 mg/kg of (A)huCD37-3 Ab, huCD37-3-SMCC-DM1, huCD37-50 Ab, huCD37-50-SMCC-DM1 or (B)huCD37-38 Ab, huCD37-38-SMCC-DM1, huCD37-56 Ab, huCD37-56-SMCC-DM1. Themean tumor volume of the different treatment groups is plotted againsttime post tumor cell inoculation.

FIG. 26 depicts results from an established xenograft study using BJABlymphoma cells implanted subcutaneous into SCID mice. Animals weretreated once on day 9 post cell inoculation with either 10 mg/kg ofhuCD37-3 Ab, huCD37-3-SMCC-DM1, huCD37-3-sulfo-mal-DM4 or 5 mg/kg ofhuCD37-3-SPP-DM1. The mean tumor volume of the different treatmentgroups is plotted against time post tumor cell inoculation.

FIG. 27 depicts results from an established xenograft model usingSU-DHL-4 diffuse large B-cell lymphoma cells implanted subcutaneous intoSCID mice. Animals were treated once on day 17 post cell inoculationwith either 10 mg/kg of huCD37-3 Ab, huCD37-3-SMCC-DM1,huCD37-3-sulfo-mal-DM4 or 5 mg/kg of huCD37-3-SPP-DM1. The median tumorvolume of the different treatment groups is plotted against time posttumor cell inoculation.

FIG. 28 depicts the results of an established xenograft model using BJABlymphoma cells implanted subcutaneous into SCID mice. Animals weretreated once on day 9 post cell inoculation with either 10 mg/kg ofhuCD37-3 Ab, huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1. The mean tumorvolume of the different treatment groups is plotted against time posttumor cell inoculation.

FIG. 29 depicts the results of an established xenograft model usingSU-DHL-4 diffuse large B-cell lymphoma cells implanted subcutaneous intoSCID mice. Animals were treated once on day 15 post cell inoculationwith either 10 mg/kg of huCD37-3 Ab, huCD37-3-SMCC-DM1 orhuCD37-3-PEG4-mal-DM1. The mean tumor volume of the different treatmentgroups is plotted against time post tumor cell inoculation.

FIG. 30 depicts the results of an assay using an established xenograftmodel with DoHH2 follicular B-cell lymphoma cells implantedsubcutaneously into SCID mice. Animals were treated starting on day 12post inoculation with (i) a single dose of 10 mg/kg of huCD37-3antibody, (ii) a single dose of 10 mg/kg of huCD37-3-SMCC-DM1 conjugate,(iii) six doses of 2 mg/kg of Rituximab twice per week for three weeks,(iv) a regimen of a single 40 mg/kg dose of cyclophosphamide and 0.5mg/kg of vincristine, along with five daily 0.2 mg/kg doses ofprednisone (CVP), or (v) a vehicle control. The median tumor volume ofthe different treatment groups is plotted against time post tumor cellinoculation.

FIG. 31 depicts the results of an assay an using established xenograftmodel with JVM3 CLL cells implanted subcutaneous into SCID mice. Animalswere treated starting on day 7 post inoculation with (i) a single doseof 10 mg/kg of huCD37-3 antibody, (ii) a 5 mg/kg dose ofhuCD37-3-SMCC-DM1 conjugate, (iii) a 10 mg/kg dose of huCD37-3-SMCC-DM1conjugate, (iv) six doses of 5 mg/kg of ofatumumab twice per week forthree weeks, (v) a single 50 mg/kg dose of bendamustine, or (vi) avehicle control. The median tumor volume of the different treatmentgroups is plotted against time post tumor cell inoculation.

FIG. 32 depicts the CD37 and CD20 expression levels measured in variousNHL and CLL tumor cell lines (A) and the in vitro cytotoxicity ofhuCD37-3-SMCC-DM1 measured in these cell lines (B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new class of CD37 binding moleculeshaving high potency in the following three cytotoxic activities againstCD37 expressing (e.g., positive) cells: induction of apoptosis, ADCC,and CDC. Further, immunoconjugates of anti-CD37 antibodies kill CD37expressing cells unexpectedly well, as demonstrated using in vivo tumormodels.

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term CD37 as used herein, refers to any native CD37, unlessotherwise indicated. CD37 is also referred to as GP52-40, leukocyteantigen CD37, and Tetraspanin-26. The term “CD37” encompasses“full-length,” unprocessed CD37 as well as any form of CD37 that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of CD37, e.g., splice variants, allelic variants, andisoforms. The CD37 polypeptides described herein can be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds, such as CD37. Insome embodiments, blocking antibodies or antagonist antibodiessubstantially or completely inhibit the biological activity of theantigen. The biological activity can be reduced by 10%, 20%, 30%, 50%,70%, 80%, 90%, 95%, or even 100%.

The term “anti-CD37 antibody” or “an antibody that binds to CD37” refersto an antibody that is capable of binding CD37 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD37. The extent of binding of an anti-CD37 antibodyto an unrelated, non-CD37 protein can be less than about 10% of thebinding of the antibody to CD37 as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antibody that binds to CD37 has adissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g. murine)antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)).

The amino acid position numbering as in Kabat, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence can containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain can include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues can be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Chothiarefers instead to the location of the structural loops (Chothia and LeskJ. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loopwhen numbered using the Kabat numbering convention varies between H32and H34 depending on the length of the loop (this is because the Kabatnumbering scheme places the insertions at H35A and H35B; if neither 35Anor 35B is present, the loop ends at 32; if only 35A is present, theloop ends at 33; if both 35A and 35B are present, the loop ends at 34).The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical Kd value. For example, an antibody which has an affinity foran antigen of “0.6 nM or better”, the antibody's affinity for theantigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any valueless than 0.6 nM.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

An antibody is said to “competitively inhibit” binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values can be less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, or less than about 10% as a function ofthe value for the reference/comparator antibody.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cell or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,an antibody, polynucleotide, vector, cell, or composition which isisolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The term “immunoconjugate” or “conjugate” as used herein refers to acompound or a derivative thereof that is linked to a cell binding agent(i.e., an anti-CD37 antibody or fragment thereof) and is defined by ageneric formula: C-L-A, wherein C=cytotoxin, L=linker, and A=cellbinding agent or anti-CD37 antibody or antibody fragment.Immunoconjugates can also be defined by the generic formula in reverseorder: A-L-C.

A “linker” is any chemical moiety that is capable of linking a compound,usually a drug, such as a maytansinoid, to a cell-binding agent such asan anti CD37 antibody or a fragment thereof in a stable, covalentmanner. Linkers can be susceptible to or be substantially resistant toacid-induced cleavage, light-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Suitable linkers are well known in the art and include, for example,disulfide groups, thioether groups, acid labile groups, photolabilegroups, peptidase labile groups and esterase labile groups. Linkers alsoinclude charged linkers, and hydrophilic forms thereof as describedherein and know in the art.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. “Tumor” and “neoplasm” refer to one or more cells that resultfrom excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.Examples of “cancer” or “tumorigenic” diseases which can be treatedand/or prevented include B-cell lymphomas including NHL, precursorB-cell lymphoblastic leukemia/lymphoma and mature B-cell neoplasms, suchas B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma(SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma,mantle cell lymphoma (MCL), follicular lymphoma (FL), includinglow-grade, intermediate-grade and high-grade FL, cutaneous folliclecenter lymphoma, marginal zone B-cell lymphoma (MALT type, nodal andsplenic type), hairy cell leukemia, diffuse large B-cell lymphoma,Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplantlymphoproliferative disorder, Waldenstrom's macroglobulinemia, andanaplastic large-cell lymphoma (ALCL).

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. The formulation can be sterile.

An “effective amount” of an antibody as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” can be determined empirically and in a routine manner, inrelation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug can reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent or stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentor stop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. See the definition herein of “treating”. To the extent the drugcan prevent growth and/or kill existing cancer cells, it can becytostatic and/or cytotoxic. A “prophylactically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result. Typically but notnecessarily, since a prophylactic dose is used in subjects prior to orat an earlier stage of disease, the prophylactically effective amountwill be less than the therapeutically effective amount.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label can be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Chemotherapeuticagents include, for example, antagonists of CD20 such as Rituximab andcyclophosphamide, doxorubicin, vincristine, prednisone, fludarabine,etoposide, methotrexate, lenalidomide, chlorambucil, bentamustine and/ormodified versions of such chemotherapeutics.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorgenic frequency, or tumorgeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; or some combination of effects.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidecan comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure canbe imparted before or after assembly of the polymer. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars can be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, orcan be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls can also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,.alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages can be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

The term “vector” means a construct, which is capable of delivering, andoptionally expressing, one or more gene(s) or sequence(s) of interest ina host cell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified inKarlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, andincorporated into the NBLAST and XBLAST programs (Altschul et al., 1991,Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLASTcan be used as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) can be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity can be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Identity can exist over a region of the sequences that is at least about10, about 20, about 40-60 residues in length or any integral valuetherebetween, and can be over a longer region than 60-80 residues, forexample, at least about 90-100 residues, and in some embodiments, thesequences are substantially identical over the full length of thesequences being compared, such as the coding region of a nucleotidesequence for example.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In someembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the invention do not abrogate the bindingof the polypeptide or antibody containing the amino acid sequence, tothe antigen(s), i.e., the CD37 to which the polypeptide or antibodybinds. Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen binding are well-known inthe art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993);Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al.Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

II. CD37 Binding Agents

The present invention provides agents that specifically bind CD37. Theseagents are referred to herein as “CD37 binding agents.” The full-lengthamino acid sequences for human, macaca, and murine CD37 are known in theart and also provided herein as represented by SEQ ID NOs:1-3,respectively.

Human CD37: (SEQ ID NO: 1)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLAYR Macaca CD37: (SEQ ID NO: 2)MSAQESCLSLIKYFLFVFNLFFFVILGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGVFTMGLALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLQDIVEKTIQRYHTNPEETAAEESWDYVQFQLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLRYR Murine CD37 (NP_031671): (SEQ ID NO: 3)MSAQESCLSLIKYFLFVFNLFFFVLGGLIFCFGTWILIDKTSFVSFVGLSFVPLQTWSKVLAVSGVLTMALALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPFVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNIISIVGICLGVGLLELGFMTLSIFLCRNLDHVYDRLARYR

In certain embodiments, the CD37 binding agents are antibodies,immunoconjugates or polypeptides. In some embodiments, the CD37 bindingagents are humanized antibodies.

In certain embodiments, the CD37-binding agents have one or more of thefollowing effects: inhibit proliferation of tumor cells, reduce thetumorigenicity of a tumor by reducing the frequency of cancer stem cellsin the tumor, inhibit tumor growth, increase survival, trigger celldeath of tumor cells, differentiate tumorigenic cells to anon-tumorigenic state, or prevent metastasis of tumor cells.

In certain embodiments, the CD37-binding agents are capable of inducingcomplement dependent cytotoxicity. For example, treatment of cells withthe CD37-binding agents can result in CDC activity that reduces cellviability to less than about 80%, less than about 70%, less than about60%, less than about 50%, less than about 40% or less than about 35% ofthe cell viability of untreated cells. Treatment of cells with theCD37-binding agents can also result in CDC activity that reduces cellviability to about 70-80%, about 60-70%, about 50-60%, about 40-50%, orabout 30-40% of the cell viability of untreated cells. In someparticular embodiments, the CD37-binding agents are capable of inducingcomplement dependent cytotoxicity in Ramos cells.

In certain embodiments, the CD37-binding agents are capable of inducingantibody dependent cell mediated cytotoxicity (ADCC). For example,treatment of cells with the CD37-binding agents can result in ADCCactivity that produces at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 60% celllysis. Treatment of cells with the CD37-binding agents can result inADCC activity that produces about 10-20%, about 20-30%, about 30-40%, orabout 40-50% cell lysis. Treatment of cells with the CD37-binding agentscan also result in ADCC activity that produces about 10-50%, about20-50%, about 30-50%, or about 40-50% cell lysis. In some particularembodiments, the CD37-binding agents are capable of inducing ADCC inDaudi, Ramos, and/or Granata-519 cells.

In some embodiments, the CD37-binding agents are capable of inducingapoptosis. For example, treatment of cells with the CD37-binding agentscan induce apoptosis in at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 55% of cells.In some particular embodiments, the CD37-binding agents are capable ofinducing apoptosis in Ramos cells and/or Raji cells.

In some embodiments, the CD37-binding agents are capable of reducingtumor volume. The ability of a CD37-binding agent to reduce tumor volumecan be assessed, for example, by measuring a % T/C value, which is themedian tumor volume of treated subjects divided by the median tumorvolume of the control subjects. In some embodiments, treatment with aCD37-binding agent results in a % T/C value that is less than about 55%,less than about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, or less than about 5%. Insome particular embodiments, the CD37-binding agents can reduce tumorsize in a BJAB xenograft model and/or a SU-DHL-4 xenograft model.

In certain embodiments, immunoconjugates or other agents thatspecifically bind human CD37 trigger cell death via a cytotoxic agent.For example, in certain embodiments, an antibody to a human CD37antibody is conjugated to a maytansinoid that is activated in tumorcells expressing the CD37 by protein internalization. In certainalternative embodiments, the agent or antibody is not conjugated.

In certain embodiments, the CD37-binding agents are capable ofinhibiting tumor growth. In certain embodiments, the CD37-binding agentsare capable of inhibiting tumor growth in vivo (e.g., in a xenograftmouse model and/or in a human having cancer).

The CD37-binding agents include CD37 antibodies CD37-3, CD37-12,CD37-38, CD37-50, CD37-51, CD37-56 and CD37-57 and fragments, variantsand derivatives thereof. The CD37-binding agents also includeCD37-binding agents that specifically bind to the same CD37 epitope asan antibody selected from the group consisting of CD37-3, CD37-12,CD37-38, CD37-50, CD37-51, CD37-56 and CD37-57. The CD37-binding agentsalso include CD37-binding agents that competitively inhibit an antibodyselected from the group consisting of CD37-3, CD37-12, CD37-38, CD37-50,CD37-51, CD37-56 and CD37-57.

In some particular embodiments, the binding of the CD37-binding agentsto CD37 does not require human CD37 amino acids 109-138. Thus, someCD37-binding agents bind to a polypeptide comprising the amino acidsequence of SEQ ID NO:180. In other embodiments, the binding of theCD37-binding agents to CD37 is disrupted by mutation of human CD37 aminoacids 202-243. Thus, some CD37-binding agents do not bind to apolypeptide comprising the amino acid sequence of SEQ ID NO:184.

In some embodiments, the CD37-binding agents bind to a polypeptide ofSEQ ID NO:180 and to a polypeptide of SEQ ID NO:183, but do not bind toa polypeptide of SEQ ID NO:184.

In some embodiments, the CD37-binding agents bind to a polypeptide ofSEQ ID NO:190. In some embodiments, the CD37-binding agents bind to apolypeptide of SEQ ID NO:190 and a polypeptide of SEQ ID NO:189. In someembodiments, the CD37-binding agents bind to a polypeptide of SEQ IDNO:190 and a polypeptide of SEQ ID NO:188.

In some embodiments, the CD37-binding agent binds to a polypeptide ofSEQ ID NO:192, but does not bind to a polypeptide of SEQ ID NO:194. Insome embodiments, the CD37-binding agent binds to a polypeptide of SEQID NO:193, but does not bind to a polypeptide of SEQ ID NO:194.

CD37 peptide fragments to which certain CD37-binding agents bind toinclude, but are not limited to, CD37 fragments comprising, consistingessentially of, or consisting of amino acids 200-243 of SEQ ID NO: 1,amino acids 202-220 or SEQ ID NO:1, or amino acids 221-243 of SEQ IDNO: 1. In some embodiments, the CD37-binding agent is specifically bindsto a human CD37 epitope comprising amino acids 202-243 of SEQ ID NO:1.In some embodiments, the binding of the CD37-binding agent to CD37requires amino acids 202-243 of SEQ ID NO:1. In some embodiments, thebinding of the CD37-binding agent to CD37 requires amino acids 200-220of SEQ ID NO: 1. In some embodiments, the binding of the CD37-bindingagent to CD37 requires amino acids 221-243 of SEQ ID NO:1.

The CD37-binding agents also include CD37-binding agents that comprisethe heavy and light chain CDR sequences of CD37-3, CD37-12, CD37-38,CD37-50, CD37-51, CD37-56 or CD37-57. The heavy and light chain CDRs ofCD37-38, CD37-50, CD37-51, CD37-56 and CD37-57 contain relatedsequences. Therefore, the CD-37 binding agents can also comprise heavyand light chain CDR sequences that comprise a consensus sequenceobtained by the alignment of CD37-38, CD37-50, CD37-51, CD37-56 andCD37-57. The CDR sequences of CD37-3, CD37-12, CD37-38, CD37-50,CD37-51, CD37-56 and CD37-57, as well as the consensus sequence ofCD37-38, CD37-50, CD37-51, CD37-56 and CD37-57 are described in Tables 1and 2 below.

TABLE 1 Variable heavy chain CDR amino acid sequences Antibody VH-CDR1VH-CDR2 VH-CDR3 CD37-3 TSGVS VIWGDGSTN GGYSLAH (SEQ ID (SEQ ID NO: 5)(SEQ ID NO: 6) NO: 4) CD37-12 KYGMN WINTNTGESR GTVVAD (SEQ ID(SEQ ID NO: 8) (SEQ ID NO: 9) NO: 7) CD37-38 SGFGWH YILYSGGTDGYYGYGAWFVY (SEQ ID (SEQ ID NO: 11) (SEQ ID NO: 12) NO: 10) CD37-50SGFAWH YILYSGSTV GYYGYGAWFAY (SEQ ID (SEQ ID NO: 14) (SEQ ID NO: 15)NO: 13) CD37-51 SGFAWH YIHYSGSTN GYYGFGAWFVY (SEQ ID (SEQ ID NO: 17)(SEQ ID NO: 18) NO: 16) CD37-56 SGFAWH YIHYSGGTN GYYGFGAWFAY (SEQ ID(SEQ ID NO: 20) (SEQ ID NO: 21) NO: 19) CD37-57 SGFAWH YILYSGSTVGYYGYGAWFAY (SEQ ID (SEQ ID NO: 23) (SEQ ID NO: 24) NO: 22) CONSENSUSSGF[A or YI[L or H]YSG[G GYYG[Y or F]GAW G]WH or S]T[D, V, or F[V or A]Y(SEQ ID N] (SEQ ID NO: 27) NO: 25) (SEQ ID NO: 26)

TABLE 2 Variable light chain CDR amino acid sequences Antibody VL-CDR1VL-CDR2 VL-CDR3 CD37-3 RASENIRSNLA VATNLAD QHYWGTTWT (SEQ ID(SEQ ID NO: 29) (SEQ ID NO: 30) NO: 28) CD37-12 RASQSVSTSSY YASNLASQHSWEIPYT SYLY (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 31) CD37-38SASSSVTYMH DTSKLAS QQWISNPPT (SEQ ID (SEQ ID NO: 35) (SEQ ID NO: 36)NO: 34) CD37-50 SATSSVTYMH DTSKLPY QQWSDNPPT (SEQ ID (SEQ ID NO: 38)(SEQ ID NO: 39) NO: 37) Humanized DTSNLPY (SEQ ID NO: 40) CD37-51SATSSVTYMH DTSKLAS QQWSSNPPT (SEQ ID (SEQ ID NO: 42) (SEQ ID NO: 43)NO: 41) CD37-56 SASSSVTYMH DTSKLAS QQWISDPPT (SEQ ID (SEQ ID NO: 45)(SEQ ID NO: 46) NO: 44) Humanized DTSNLAS (SEQ ID NO: 47) CD37-57SATSSVTYMH DTSKLAS QQWSDNPPT (SEQ ID (SEQ ID NO: 49) (SEQ ID NO: 50)NO: 48) Humanized DTSNLAS (SEQ ID NO: 51) CONSENSUS SA[T or S]DTS[K or N]L[A QQW[I or S] SSVTYMH or P][S or Y] [S or D][N or (SEQ ID(SEQ ID NO: 53) D]PPT NO: 52) (SEQ ID NO: 54)

The CD37 binding molecules can be antibodies or antigen bindingfragments that specifically bind to CD37 that comprise the CDRs ofCD37-3, CD37-12, CD37-50, CD37-51, CD37-56, or CD37-57 with up to four(i.e. 0, 1, 2, 3, or 4) conservative amino acid substitutions per CDR.

Polypeptides an comprise one of the individual variable light chains orvariable heavy chains described herein. Antibodies and polypeptides canalso comprise both a variable light chain and a variable heavy chain.The variable light chain and variable heavy chain sequences of murine,chimeric, and humanized CD37-3, CD37-12, CD37-50, CD37-51, CD37-56, andCD37-57 antibodies are provided in Tables 3 and 4 below.

TABLE 3 Variable heavy chain amino acid sequences AntibodyVH Amino Acid Sequence (SEQ ID NO) muCD37-3QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVS WVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHW GQGTLVTVSA (SEQ ID NO: 55) chCD37-3QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVS WVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHW GQGTLVTVSA (SEQ ID NO: 56)huCD37-3v1.0 QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHPSLKSRLSIK KDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHWGQGTLVTVSS (SEQ ID NO: 57) huCD37-3v1.1QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVS WVRQPPGKGLEWLGVIWGDGSTNYHSSLKSRLSIKKDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHW GQGTLVTVSS (SEQ ID NO: 58) muCD37-12QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMN WVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAFSLETSASTAYLQINNLKYEDTATYFCGRGTVVADW GQGTTLTVSS (SEQ ID NO: 59) chCD37-12QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMN WVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAFSLETSASTAYLQINNLKYEDTATYFCGRGTVVADW GQGTTLTVSS (SEQ ID NO: 60) muCD37-38DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGW HWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGA WFVYWGQGTLVTVSA (SEQ ID NO: 61)chCD37-38 QVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGWHWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISI TRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGAWFVYWGQGTLVTVSA (SEQ ID NO: 62) huCD37-38QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFGW HWIRQFPGKGLEWMAYILYSGGTDYNPSLKSRISITRDTSKNQFFLRLSSVTAADTATYYCARGYYGYGA WFVYWGQGTLVTVSS (SEQ ID NO: 63)muCD37-50 DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISI TRDTSKNHFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSA (SEQ ID NO: 64) huCD37-50QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAW HWIRQHPGNKLEWMGYILYSGSTVYSPSLKSRISITRDTSKNHFFLQLNSVTAADTATYYCARGYYGYGA WFAYWGQGTLVTVSA (SEQ ID NO: 65)muCD37-51 DVQLQESGPDLLKPSQSLSLTCTVTGYSISSGFAWHWIRQFPGNKLEWMGYIHYSGSTNYSPSLKSRISI TRDSSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFVYWGQGTLVTVSA (SEQ ID NO: 66) huCD37-51EVQLVESGPEVLKPGESLSLTCTVSGYSISSGFAW HWIRQFPGKGLEWMGYIHYSGSTNYSPSLQGRISITRDSSINQFFLQLNSVTASDTATYYCARGYYGFGA WFVYWGQGTLVTVSA (SEQ ID NO: 67)muCD37-56 DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRVSI TRDTSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFAYWGQGTLVPVSA (SEQ ID NO: 68) huCD37-56QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFAW HWIRQFPGKGLEWMGYIHYSGGTNYNPSLKSRVSITRDTSKNQFFLQLNSVTAADTATYYCARGYYGFGA WFAYWGQGTLVPVSA (SEQ ID NO: 69)muCD37-57 DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISI TRDTSKNQFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSA (SEQ ID NO: 70) huCD37-57QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAW HWIRQFPGKGLEWMGYILYSGSTVYSPSLKSRISITRDTSKNQFFLQLNSVTAADTATYYCARGYYGYGA WFAYWGQGTLVTVSA (SEQ ID NO: 71)

TABLE 4 Variable light chain amino acid sequences AntibodyVL Amino Acid Sequence (SEQ ID NO) muCD37-3DIQMTQSPASLSVSVGETVTITCRASENIRSNLA WYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGT KLEIKR (SEQ ID NO: 72) chCD37-3DIQMTQSPASLSVSVGETVTITCRASENIRSNLA WYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGT KLEIKR (SEQ ID NO: 73) huCD37-3DIQMTQSPSSLSVSVGERVTITCRASENIRSNLA (1.0 and 1.1)WYQQKPGKSPKLLVNVATNLADGVPSRFSGSGTD YSLKINSLQPEDFGTYYCQHYWGTTWTFGQGTKLEIKR (SEQ ID NO: 74) muCD37-12 DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSG SGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKR (SEQ ID NO: 75) chCD37-12 DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSG SGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKR (SEQ ID NO: 76) muCD37-38 QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGT SYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKR (SEQ ID NO: 77) chCD37-38 QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGT SYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKR (SEQ ID NO: 78) huCD37-38 DIVLTQSPASMSASPGERVTMTCSASSSVTYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKR (SEQ ID NO: 79) muCD37-50 QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLPYGVPGRFSGSGSGT SYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKR (SEQ ID NO: 80) huCD37-50 EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSNLPYGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKR (SEQ ID NO: 81) muCD37-51 QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGT SYSLTISNMEAEDAATYYCQQWSSNPPTFGSGTKLEIKR (SEQ ID NO: 82) huCD37-51 EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSKLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQQWSSNPPTFGQGTKLEIKR (SEQ ID NO: 83) muCD37-56 QIVLTQSPAFMSASPGDKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGT SYSLTISTMEAEDAATYYCQQWISDPPTFGGGTKLEIKR (SEQ ID NO: 84) huCD37-56 DIVLTQSPAFMSASPGEKVTMTCSASSSVTYMHWYQQKPDQSPKRWIYDTSNLASGVPSRFSGGGSGT DYSLTISSMEAEDAATYYCQQWISDPPTFGQGTKLEIKR (SEQ ID NO: 85) muCD37-57 QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKR (SEQ ID NO: 86) huCD37-57 EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPRRWIYDTSNLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKR (SEQ ID NO: 87)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:55-71; and/or (b) apolypeptide having at least about 90% sequence identity to SEQ IDNOs:72-87. In certain embodiments, the polypeptide comprises apolypeptide having at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% sequence identityto SEQ ID NOs:55-87. Thus, in certain embodiments, the polypeptidecomprises (a) a polypeptide having at least about 95% sequence identityto SEQ ID NOs:55-71, and/or (b) a polypeptide having at least about 95%sequence identity to SEQ ID NOs:72-87. In certain embodiments, thepolypeptide comprises (a) a polypeptide having the amino acid sequenceof SEQ ID NOs:55-71; and/or (b) a polypeptide having the amino acidsequence of SEQ ID NOs:72-87. In certain embodiments, the polypeptide isan antibody and/or the polypeptide specifically binds CD37. In certainembodiments, the polypeptide is a murine, chimeric, or humanizedantibody that specifically binds CD37. In certain embodiments, thepolypeptide having a certain percentage of sequence identity to SEQ IDNOs:55-87 differs from SEQ ID NOs:55-87 by conservative amino acidsubstitutions only.

Polypeptides can comprise one of the individual light chains or heavychains described herein. Antibodies and polypeptides can also compriseboth a light chain and a heavy chain. The light chain and variable chainsequences of murine, chimeric, and humanized CD37-3, CD37-12, CD37-50,CD37-51, CD37-56, and CD37-57 antibodies are provided in Tables 5 and 6below.

TABLE 5 Full-length heavy chain amino acid sequencesFull-Length Heavy Chain Amino Acid Antibody Sequence (SEQ ID NO)muCD37-3 QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIK KDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTL GCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS YSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 88) chCD37-3 QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALKSRLSIK KDHSKSQVFLKLNSLQTDDTATYYCAKGGYSLAHWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 89) huCD37-3v1.0 QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHPSLKSRLSIK KDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 90) huCD37-3v1.1 QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKGLEWLGVIWGDGSTNYHSSLKSRLSIK KDHSKSQVFLKLNSLTAADTATYYCAKGGYSLAHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 91) muCD37-12 QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMNWVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAF SLETSASTAYLQINNLKYEDTATYFCGRGTVVADWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTL GCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS YSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 92) chCD37-12 QIQLVQSGPELKKPGETVKISCKASGYTFTKYGMNWVKQAQGKGLKWMGWINTNTGESRNAEEFKGRFAF SLETSASTAYLQINNLKYEDTATYFCGRGTVVADWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 93) muCD37 -38 DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGWHWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISI TRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGAWFVYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTN SMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASS TKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVE VHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPP PKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTF TCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO: 94) chCD37-38 QVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFGWHWIRQFPGNKLEWMAYILYSGGTDYNPSLKSRISI TRDTSKNQFFLRLSSVTTEDTATYYCARGYYGYGAWFVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 95) huCD37-38 QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFGWHWIRQFPGKGLEWMAYILYSGGTDYNPSLKSRISI TRDTSKNQFFLRLSSVTAADTATYYCARGYYGYGAWFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 96) muCD37-50 DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISI TRDTSKNHFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTG SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS TKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQ VYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNW VERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 97) huCD37-50 QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAWHWIRQHPGNKLEWMGYILYSGSTVYSPSLKSRISI TRDTSKNHFFLQLNSVTAADTATYYCARGYYGYGAWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 98) muCD37-51 DVQLQESGPDLLKPSQSLSLTCTVTGYSISSGFAWHWIRQFPGNKLEWMGYIHYSGSTNYSPSLKSRISI TRDSSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFVYVVGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPAS STKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQIS WFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP QVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKN WVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 99) huCD37-51 EVQLVESGPEVLKPGESLSLTCTVSGYSISSGFAWHWIRQFPGKGLEWMGYIHYSGSTNYSPSLQGRISI TRDSSINQFFLQLNSVTASDTATYYCARGYYGFGAWFVYVVGQGTLVTVSAASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 100) muCD37-56 DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYIHYSGGTNYNPSLKSRVSI TRDTSKNQFFLQLNSVTTEDTATYYCARGYYGFGAWFAYWGQGTLVPVSAAKTTPPSVYPLAPGSAAQTN SMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSMRPSETVTCNVAHPASS TKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVE VHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPP PKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTF TCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO: 101) huCD37-56 QVQLQESGPGLVKPSQSLSLTCTVSGYSITSGFAWHWIRQFPGKGLEWMGYIHYSGGTNYNPSLKSRVSI TRDTSKNQFFLQLNSVTAADTATYYCARGYYGFGAWFAYWGQGTLVPVSAASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 102) muCD37-57 DVQLQESGPDLLKPSQSLSLTCTVTGYSITSGFAWHWIRQFPGNKLEWMGYILYSGSTVYSPSLKSRISI TRDTSKNQFFLQLNSVTTEDTATYYCARGYYGYGAWFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTG SSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASS TKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQ VYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNW VERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO: 103) huCD37-57 QVQLQESGPGLLKPSQSLSLTCTVSGYSITSGFAWHWIRQFPGKGLEWMGYILYSGSTVYSPSLKSRISI TRDTSKNQFFLQLNSVTAADTATYYCARGYYGYGAWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 104)

TABLE 6 Full-length light chain amino acid sequencesFull-length Light Chain Amino Acid Sequence Antibody (SEQ ID NO)muCD37-3 DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC(SEQ ID NO: 105) chCD37-3DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLLVNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWGTTWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC(SEQ ID NO: 106) huCD37-3DIQMTQSPSSLSVSVGERVTITCRASENIRSNLAWYQQKPGKSPKLLVNVAT (1.0 and 1.1)NLADGVPSRFSGSGSGTDYSLKINSLQPEDFGTYYCQHYWGTTWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC(SEQ ID NO: 107) muCD37-12DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI VKSFNRNEC(SEQ ID NO: 108) chCD37-12DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYLYWFQQKPGQPPKLLIKYASNLASGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC(SEQ ID NO: 109) muCD37-38QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC(SEQ ID NO: 110) chCD37-38QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(SEQ ID NO: 111) huCD37-38DIVLTQSPASMSASPGERVTMTCSASSSVTYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWISNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(SEQ ID NO: 112) muCD37-50QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLPYGVPGRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC(SEQ ID NO: 113) huCD37-50EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSNLPYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(SEQ ID NO: 114) muCD37-51QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISNMEAEDAATYYCQQWSSNPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC(SEQ ID NO: 115) huCD37-51EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(SEQ ID NO: 116) muCD37-56QIVLTQSPAFMSASPGDKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGGGSGTSYSLTISTMEAEDAATYYCQQWISDPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC(SEQ ID NO: 117) huCD37-56DIVLTQSPAFMSASPGEKVTMTCSASSSVTYMHWYQQKPDQSPKRWIYDTSNLASGVPSRFSGGGSGTDYSLTISSMEAEDAATYYCQQWISDPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(SEQ ID NO: 118) muCD37-57QIVLTQSPAIMSASPGEKVTMTCSATSSVTYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC(SEQ ID NO: 119) huCD37-57EIVLTQSPATMSASPGERVTMTCSATSSVTYMHWYQQKPGQSPRRWIYDTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSDNPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC(SEQ ID NO: 120)

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 90% sequence identity to SEQ ID NOs:88-104; and/or (b) apolypeptide having at least about 90% sequence identity to SEQ IDNOs:105-120. In certain embodiments, the polypeptide comprises apolypeptide having at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% sequence identityto SEQ ID NOs:88-120. Thus, in certain embodiments, the polypeptidecomprises (a) a polypeptide having at least about 95% sequence identityto SEQ ID NOs:88-104, and/or (b) a polypeptide having at least about 95%sequence identity to SEQ ID NOs:105-120. In certain embodiments, thepolypeptide comprises (a) a polypeptide having the amino acid sequenceof SEQ ID NOs:88-104; and/or (b) a polypeptide having the amino acidsequence of SEQ ID NOs:105-120. In certain embodiments, the polypeptideis an antibody and/or the polypeptide specifically binds CD37. Incertain embodiments, the polypeptide is a murine, chimeric, or humanizedantibody that specifically binds CD37. In certain embodiments, thepolypeptide having a certain percentage of sequence identity to SEQ IDNOs:88-120 differs from SEQ ID NOs:88-120 by conservative amino acidsubstitutions only.

In certain embodiments, the CD37 antibody can be the antibody producedfrom a hybridoma selected from the group consisting of consisting ofATCC Deposit Designation PTA-10664, deposited with the ATCC on Feb. 18,2010, ATCC Deposit Designation PTA-10665, deposited with the ATCC onFeb. 18, 2010, ATCC Deposit Designation PTA-10666, deposited with theATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10667 deposited withthe ATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10668, depositedwith the ATCC on Feb. 18, 2010, ATCC Deposit Designation PTA-10669,deposited with the ATCC on Feb. 18, 2010, and ATCC Deposit DesignationPTA-10670, deposited with the ATCC on Feb. 18, 2010. In certainembodiments, the antibody comprises the VH-CDRs and the VL-CDRS of theantibody produced from a hydridoma selected from the group consisting ofPTA-10665, PTA-10666, PTA-10667, PTA-10668, PTA-10669, and PTA-10670.

In certain embodiments, the CD37 antibody can comprise a light chainencoded by the recombinant plasmid DNA phuCD37-3LC (ATCC DepositDesignation PTA-10722, deposited with the ATCC on Mar. 18, 2010). Incertain embodiments, the CD37 antibody can comprise a heavy chainencoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (ATCC DepositDesignation PTA-10723, deposited with the ATCC on Mar. 18, 2010). Incertain embodiments, the CD37 antibody can comprise a light chainencoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722) and aheavy chain encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0(PTA-10723). In certain embodiments, the CD37 antibody can comprise theVL-CDRs encoded by the recombinant plasmid DNA phuCD37-3LC (PTA-10722)and the VH-CDRs encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0(PTA-10723).

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Lymphocytes can also be immunized in vitro. Following immunization, thelymphocytes are isolated and fused with a suitable myeloma cell lineusing, for example, polyethylene glycol, to form hybridoma cells thatcan then be selected away from unfused lymphocytes and myeloma cells.Hybridomas that produce monoclonal antibodies directed specificallyagainst a chosen antigen as determined by immunoprecipitation,immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay(RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagatedeither in vitro culture using standard methods (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in vivo asascites tumors in an animal. The monoclonal antibodies can then bepurified from the culture medium or ascites fluid as described forpolyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the human CD37 is ahumanized antibody. In certain embodiments, such antibodies are usedtherapeutically to reduce antigenicity and HAMA (human anti-mouseantibody) responses when administered to a human subject. Humanizedantibodies can be produced using various techniques known in the art. Incertain alternative embodiments, the antibody to CD37 is a humanantibody.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, e.g., Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No.5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies, asdescribed, for example, in Vaughan et al., 1996, Nat. Biotech.,14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162,Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al.,1991, J. Mol. Biol., 222:581). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which isincorporated by reference in its entirety). Affinity maturationstrategies and chain shuffling strategies (Marks et al., 1992,Bio/Technology 10:779-783, incorporated by reference in its entirety)are known in the art and can be employed to generate high affinity humanantibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize a CD37. Bispecific antibodies are antibodies that are capableof specifically recognizing and binding at least two different epitopes.The different epitopes can either be within the same molecule (e.g. thesame CD37) or on different molecules such that both, for example, theantibodies can specifically recognize and bind a CD37 as well as, forexample, 1) an effector molecule on a leukocyte such as a T-cellreceptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2) acytotoxic agent as described in detail below.

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodiesare common in the art (Millstein et al., 1983, Nature 305:537-539;Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods inEnzymol. 121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalabyet al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J.Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; andU.S. Pat. No. 5,731,168). Antibodies with more than two valencies arealso contemplated. For example, trispecific antibodies can be prepared(Tutt et al., J. Immunol. 147:60 (1991)). Thus, in certain embodimentsthe antibodies to CD37 are multispecific.

In certain embodiments are provided an antibody fragment to, forexample, increase tumor penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117; Brennan et al., 1985, Science, 229:81). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to CD37 (see U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof Fab expression libraries (Huse, et al., Science 246:1275-1281 (1989))to allow rapid and effective identification of monoclonal Fab fragmentswith the desired specificity for CD37, or derivatives, fragments,analogs or homologs thereof. Antibody fragments can be produced bytechniques in the art including, but not limited to: (a) a F(ab′)2fragment produced by pepsin digestion of an antibody molecule; (b) a Fabfragment generated by reducing the disulfide bridges of an F(ab′)2fragment, (c) a Fab fragment generated by the treatment of the antibodymolecule with papain and a reducing agent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides of ahuman CD37. In this regard, the variable region can comprise or bederived from any type of mammal that can be induced to mount a humoralresponse and generate immunoglobulins against the desired tumorassociated antigen. As such, the variable region of the modifiedantibodies can be, for example, of human, murine, non-human primate(e.g. cynomolgus monkeys, macaques, etc.) or lupine origin. In someembodiments both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencechanging. Although the CDRs can be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and possibly from an antibody from adifferent species. It is not always necessary to replace all of the CDRswith the complete CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, insome cases it is only necessary to transfer those residues that arenecessary to maintain the activity of the antigen binding site. Giventhe explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the competence of those skilled in theart, either by carrying out routine experimentation or by trial anderror testing to obtain a functional antibody with reducedimmunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g., full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization or reduced serum half-life when compared with an antibodyof approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. That is, the modified antibodies disclosedherein can comprise alterations or modifications to one or more of thethree heavy chain constant domains (CH1, CH2 or CH3) and/or to the lightchain constant domain (CL). In some embodiments, modified constantregions wherein one or more domains are partially or entirely deletedare contemplated. In some embodiments, the modified antibodies willcomprise domain deleted constructs or variants wherein the entire CH2domain has been removed (ΔCH2 constructs). In some embodiments, theomitted constant region domain will be replaced by a short amino acidspacer (e.g. 10 residues) that provides some of the molecularflexibility typically imparted by the absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

In certain embodiments, the CD37-binding antibodies provide for alteredeffector functions that, in turn, affect the biological profile of theadministered antibody. For example, the deletion or inactivation(through point mutations or other means) of a constant region domain canreduce Fc receptor binding of the circulating modified antibody therebyincreasing tumor localization. In other cases, it can be that constantregion modifications, consistent with this invention, moderatecomplement binding and thus reduce the serum half life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region can be used to eliminate disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or antibody flexibility. Similarly,modifications to the constant region in accordance with this inventioncan easily be made using well known biochemical or molecular engineeringtechniques well within the purview of the skilled artisan.

In certain embodiments, a CD37-binding agent that is an antibody doesnot have one or more effector functions. For instance, in someembodiments, the antibody has no antibody-dependent cellularcytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity(CDC) activity. In certain embodiments, the antibody does not bind to anFc receptor and/or complement factors. In certain embodiments, theantibody has no effector function.

It will be noted that in certain embodiments, the modified antibodiescan be engineered to fuse the CH3 domain directly to the hinge region ofthe respective modified antibodies. In other constructs it can bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer can be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain embodiments, any spacer added tothe construct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention can be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain can be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it can be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement CLQ binding) to bemodulated. Such partial deletions of the constant regions can improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies can be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it can be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Certain embodiments can comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as decreasing or increasing effector function orprovide for more cytotoxin or carbohydrate attachment. In suchembodiments it can be desirable to insert or replicate specificsequences derived from selected constant region domains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a human CD37. It will berecognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against CD37 protein. Such mutantsinclude deletions, insertions, inversions, repeats, and typesubstitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human CD37. Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding apolypeptide chain of an anti-CD37 antibody, or fragment thereof,operatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements can include an operator sequence to control transcription. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants can additionally be incorporated. DNA regions areoperatively linked when they are functionally related to each other. Forexample, DNA for a signal peptide (secretory leader) is operativelylinked to DNA for a polypeptide if it is expressed as a precursor whichparticipates in the secretion of the polypeptide; a promoter isoperatively linked to a coding sequence if it controls the transcriptionof the sequence; or a ribosome binding site is operatively linked to acoding sequence if it is positioned so as to permit translation.Structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. Alternatively, where recombinant protein is expressedwithout a leader or transport sequence, it can include an N-terminalmethionine residue. This residue can optionally be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a CD37-binding polypeptide orantibody (or a CD37 protein to use as an antigen) include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin as describedbelow. Cell-free translation systems could also be employed. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference. Additionalinformation regarding methods of protein production, including antibodyproduction, can be found, e.g., in U.S. Patent Publication No.2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and InternationalPatent Publication No. WO 04009823, each of which is hereby incorporatedby reference herein in its entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a CD37-binding agent. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNo. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

In certain embodiments, the CD37-binding agent is a polypeptide that isnot an antibody. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See, e.g., Skerra, Curr. Opin. Biotechnol.,18:295-304 (2007), Hosse et al., Protein Science, 15:14-27 (2006), Gillet al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J.,275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each ofwhich is incorporated by reference herein in its entirety. In certainembodiments, phage display technology has been used to identify/producethe CD37-binding polypeptide. In certain embodiments, the polypeptidecomprises a protein scaffold of a type selected from the groupconsisting of protein A, a lipocalin, a fibronectin domain, an ankyrinconsensus repeat domain, and thioredoxin.

In some embodiments, the agent is a non-protein molecule. In certainembodiments, the agent is a small molecule. Combinatorial chemistrylibraries and techniques useful in the identification of non-proteinCD37-binding agents are known to those skilled in the art. See, e.g.,Kennedy et al., J. Comb. Chem, 10:345-354 (2008), Dolle et al, J. Comb.Chem., 9:855-902 (2007), and Bhattacharyya, Curr. Med. Chem., 8:1383-404(2001), each of which is incorporated by reference herein in itsentirety. In certain further embodiments, the agent is a carbohydrate, aglycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent is a nucleic acid aptamer. Aptamersare polynucleotide molecules that have been selected (e.g., from randomor mutagenized pools) on the basis of their ability to bind to anothermolecule. In some embodiments, the aptamer comprises a DNApolynucleotide. In certain alternative embodiments, the aptamercomprises an RNA polynucleotide. In certain embodiments, the aptamercomprises one or more modified nucleic acid residues. Methods ofgenerating and screening nucleic acid aptamers for binding to proteinsare well known in the art. See, e.g., U.S. Pat. No. 5,270,163, U.S. Pat.No. 5,683,867, U.S. Pat. No. 5,763,595, U.S. Pat. No. 6,344,321, U.S.Pat. No. 7,368,236, U.S. Pat. No. 5,582,981, U.S. Pat. No. 5,756,291,U.S. Pat. No. 5,840,867, U.S. Pat. No. 7,312,325, U.S. Pat. No.7,329,742, International Patent Publication No. WO 02/077262,International Patent Publication No. WO 03/070984, U.S. PatentApplication Publication No. 2005/0239134, U.S. Patent ApplicationPublication No. 2005/0124565, and U.S. Patent Application PublicationNo. 2008/0227735, each of which is incorporated by reference herein inits entirety.

III. Immunoconjugates

The present invention is also directed to conjugates (also referred toherein as immunoconjugates), comprising the anti-CD37 antibodies,antibody fragments, and their functional equivalents as disclosedherein, linked or conjugated to a drug or prodrug. Suitable drugs orprodrugs are known in the art. The drugs or prodrugs can be cytotoxicagents. The cytotoxic agent used in the cytotoxic conjugate of thepresent invention can be any compound that results in the death of acell, or induces cell death, or in some manner decreases cell viability,and includes, for example, maytansinoids and maytansinoid analogs. Othersuitable cytotoxic agents are for example benzodiazepines, taxoids,CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs,enediynes, such as calicheamicins, dolastatin and dolastatin analogsincluding auristatins, tomaymycin derivatives, leptomycin derivatives,methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, chlorambucil andmorpholino doxorubicin.

Such conjugates can be prepared by using a linking group in order tolink a drug or prodrug to the antibody or functional equivalent.Suitable linking groups are well known in the art and include, forexample, disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups and esterase labile groups.

The drug or prodrug can, for example, be linked to the anti-CD37antibody or fragment thereof through a disulfide bond. The linkermolecule or crosslinking agent comprises a reactive chemical group thatcan react with the anti-CD37 antibody or fragment thereof. The reactivechemical groups for reaction with the cell-binding agent can beN-succinimidyl esters and N-sulfosuccinimidyl esters. Additionally thelinker molecule comprises a reactive chemical group, which can be adithiopyridyl group that can react with the drug to form a disulfidebond. Linker molecules include, for example, N-succinimidyl3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al.,Biochem. J., 173: 723-737 (1978)), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Pat. No.4,563,304), N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate(sulfo-SPDB) (see US Publication No. 20090274713), N-succinimidyl4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride. For example,the antibody or cell binding agent can be modified with crosslinkingreagents and the antibody or cell binding agent containing free orprotected thiol groups thus derived is then reacted with a disulfide- orthiol-containing maytansinoid to produce conjugates. The conjugates canbe purified by chromatography, including but not limited to HPLC,size-exclusion, adsorption, ion exchange and affinity capture, dialysisor tangential flow filtration.

In another aspect of the present invention, the anti-CD37 antibody islinked to cytotoxic drugs via disulfide bonds and a polyethylene glycolspacer in enhancing the potency, solubility or the efficacy of theimmunoconjugate. Such cleavable hydrophilic linkers are described inWO2009/0134976. The additional benefit of this linker design is thedesired high monomer ratio and the minimal aggregation of theantibody-drug conjugate. Specifically contemplated in this aspect areconjugates of cell-binding agents and drugs linked via disulfide group(—S—S—) bearing polyethylene glycol spacers ((CH₂CH₂O)_(n=1-14)) with anarrow range of drug load of 2-8 are described that show relatively highpotent biological activity toward cancer cells and have the desiredbiochemical properties of high conjugation yield and high monomer ratiowith minimal protein aggregation.

Specifically contemplated in this aspect is an anti-CD37 antibody drugconjugate of formula (I) or a conjugate of formula (I′):CB—[X₁—(—CH₂—CH₂O—)_(n)—Y-D]_(m)  (I)[D-Y—(—CH₂—CH₂O—)_(n)—X₁]_(m)—CB  (I′)wherein:

CB represents an anti-CD37 antibody or fragment;

D represents a drug;

X represents an aliphatic, an aromatic or a heterocyclic unit attachedto the cell-binding agent via a thioether bond, an amide bond, acarbamate bond, or an ether bond;

Y represents an aliphatic, an aromatic or a heterocyclic unit attachedto the drug via a disulfide bond;

1 is 0 or 1;

m is an integer from 2 to 8; and

n is an integer from 1 to 24.

In some embodiments, m is an integer from 2 to 6.

In some embodiments, m is an integer from 3 to 5.

In some embodiments, n is an integer form 2 to 8. Alternatively, asdisclosed in, for example, U.S. Pat. Nos. 6,441,163 and 7,368,565, thedrug can be first modified to introduce a reactive ester suitable toreact with a cell-binding agent. Reaction of these drugs containing anactivated linker moiety with a cell-binding agent provides anothermethod of producing a cell-binding agent drug conjugate. Maytansinoidscan also be linked to anti-CD37 antibody or fragment using PEG linkinggroups, as set forth for example in U.S. Pat. No. 6,716,821. These PEGnon-cleavable linking groups are soluble both in water and innon-aqueous solvents, and can be used to join one or more cytotoxicagents to a cell binding agent. Exemplary PEG linking groups includeheterobifunctional PEG linkers that react with cytotoxic agents and cellbinding agents at opposite ends of the linkers through a functionalsulfhydryl or disulfide group at one end, and an active ester at theother end. As a general example of the synthesis of a cytotoxicconjugate using a PEG linking group, reference is again made to U.S.Pat. No. 6,716,821 which is incorporated entirely by reference herein.Synthesis begins with the reaction of one or more cytotoxic agentsbearing a reactive PEG moiety with a cell-binding agent, resulting indisplacement of the terminal active ester of each reactive PEG moiety byan amino acid residue of the cell binding agent, to yield a cytotoxicconjugate comprising one or more cytotoxic agents covalently bonded to acell binding agent through a PEG linking group. Alternatively, the cellbinding can be modified with the bifunctional PEG crosslinker tointroduce a reactive disulfide moiety (such as a pyridyldisulfide),which can then be treated with a thiol-containing maytansinoid toprovide a conjugate. In another method, the cell binding can be modifiedwith the bifunctional PEG crosslinker to introduce a thiol moiety whichcan then can be treated with a reactive disulfide-containingmaytansinoid (such as a pyridyldisulfide), to provide a conjugate.

Antibody-maytansinoid conjugates with non-cleavable links can also beprepared. Such crosslinkers are described in the art (see US PublicationNo. 20050169933) and include but are not limited to, N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC). In some embodiments,the antibody is modified with crosslinking reagents such as succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC,maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS orsuccinimidyl-iodoacetate, as described in the literature, to introduce1-10 reactive groups (Yoshitake et al, Eur. J. Biochem., 101:395-399(1979); Hashida et al, J. Applied Biochem., 56-63 (1984); and Liu et al,Biochem., 18:690-697 (1979)). The modified antibody is then reacted withthe thiol-containing maytansinoid derivative to produce a conjugate. Theconjugate can be purified by gel filtration through a Sephadex G25column or by dialysis or tangential flow filtration. The modifiedantibodies are treated with the thiol-containing maytansinoid (1 to 2molar equivalent/maleimido group) and antibody-maytansinoid conjugatesare purified by gel filtration through a Sephadex G-25 column,chromatography on a ceramic hydroxyapatite column, dialysis ortangential flow filtration or a combination of methods thereof.Typically, an average of 1-10 maytansinoids per antibody are linked. Onemethod is to modify antibodies with succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introducemaleimido groups followed by reaction of the modified antibody with athiol-containing maytansinoid to give a thioether-linked conjugate.Again conjugates with 1 to 10 drug molecules per antibody moleculeresult. Maytansinoid conjugates of antibodies, antibody fragments, andother proteins are made in the same way.

In another aspect of the invention, the CD37 antibody is linked to thedrug via a non-cleavable bond through the intermediacy of a PEG spacer.Suitable crosslinking reagents comprising hydrophilic PEG chains thatform linkers between a drug and the anti-CD37 antibody or fragment arealso well known in the art, or are commercially available (for examplefrom Quanta Biodesign, Powell, Ohio). Suitable PEG-containingcrosslinkers can also be synthesized from commercially available PEGsthemselves using standard synthetic chemistry techniques known to oneskilled in the art. The drugs can be reacted with bifunctionalPEG-containing cross linkers to give compounds of the following formula,Z—X₁—(—CH₂—CH₂O—)_(n)—Y_(p)-D, by methods described in detail in USPatent Publication 20090274713 and in WO2009/0134976, which can thenreact with the cell binding agent to provide a conjugate. Alternatively,the cell binding can be modified with the bifunctional PEG crosslinkerto introduce a thiol-reactive group (such as a maleimide orhaloacetamide) which can then be treated with a thiol-containingmaytansinoid to provide a conjugate. In another method, the cell bindingcan be modified with the bifunctional PEG crosslinker to introduce athiol moiety which can then be treated with a thiol-reactivemaytansinoid (such as a maytansinoid bearing a maleimide orhaloacetamide), to provide a conjugate.

Accordingly, another aspect of the present invention is an anti-CD37antibody drug conjugate of formula (II) or of formula (II′):CB—[X₁—(—CH₂—CH₂—O—)_(n)—Y_(p)-D]_(m)  (II)[D-Y_(p)—(—CH₂—CH₂—O—)_(n)—X₁]_(m)—CB  (II′)wherein, CB represents an anti-CD37 antibody or fragment;

D represents a drug;

X represents an aliphatic, an aromatic or a heterocyclic unit bonded tothe cell-binding agent via a thioether bond, an amide bond, a carbamatebond, or an ether bond;

Y represents an aliphatic, an aromatic, or a heterocyclic unit bonded tothe drug via a covalent bond selected from the group consisting of athioether bond, an amide bond, a carbamate bond, an ether bond, an aminebond, a carbon-carbon bond and a hydrazone bond;

1 is 0 or 1;

p is 0 or 1;

m is an integer from 2 to 15; and

n is an integer from 1 to 2000.

In some embodiments, m is an integer from 2 to 8; and

In some embodiments, n is an integer from 1 to 24.

In some embodiments, m is an integer from 2 to 6.

In some embodiments, m is an integer from 3 to 5.

In some embodiments, n is an integer from 2 to 8. Examples of suitablePEG-containing linkers include linkers having an N-succinimidyl ester orN-sulfosuccinimidyl ester moiety for reaction with the anti-CD37antibody or fragment thereof, as well as a maleimido- orhaloacetyl-based moiety for reaction with the compound. A PEG spacer canbe incorporated into any crosslinker known in the art by the methodsdescribed herein.

Many of the linkers disclosed herein are described in detail in U.S.Patent Publication Nos. 20050169933 and 20090274713, and inWO2009/0134976; the contents of which are entirely incorporated hereinby reference.

The present invention includes aspects wherein about 2 to about 8 drugmolecules (“drug load”), for example, maytansinoid, are linked to ananti-CD37 antibody or fragment thereof, the anti-tumor effect of theconjugate is much more efficacious as compared to a drug load of alesser or higher number of drugs linked to the same cell binding agent.“Drug load”, as used herein, refers to the number of drug molecules(e.g., a maytansinoid) that can be attached to a cell binding agent(e.g., an anti-CD37 antibody or fragment thereof). In one aspect, thenumber of drug molecules that can be attached to a cell binding agentcan average from about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0,8.1). N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1)and N^(2′)-deacetyl-N^(2′)-(4-mercapto-4-methyl-1-oxopentyl) maytansine(DM4) can be used.

Thus, in one aspect, an immunocongugate comprises 1 maytansinoid perantibody. In another aspect, an immunocongugate comprises 2maytansinoids per antibody. In another aspect, an immunocongugatecomprises 3 maytansinoids per antibody. In another aspect, animmunocongugate comprises 4 maytansinoids per antibody. In anotheraspect, an immunocongugate comprises 5 maytansinoids per antibody. Inanother aspect, an immunocongugate comprises 6 maytansinoids perantibody. In another aspect, an immunocongugate comprises 7maytansinoids per antibody. In another aspect, an immunocongugatecomprises 8 maytansinoids per antibody.

In one aspect, an immunoconjugate comprises about 1 to about 8maytansinoids per antibody. In another aspect, an immunoconjugatecomprises about 2 to about 7 maytansinoids per antibody. In anotheraspect, an immunoconjugate comprises about 2 to about 6 maytansinoidsper antibody. In another aspect, an immunoconjugate comprises about 2 toabout 5 maytansinoids per antibody. In another aspect, animmunoconjugate comprises about 3 to about 5 maytansinoids per antibody.In another aspect, an immunoconjugate comprises about 3 to about 4maytansinoids per antibody.

In one aspect, a composition comprising immunoconjugates has an averageof about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1) drugmolecules (e.g., maytansinoids) attached per antibody. In one aspect, acomposition comprising immunoconjugates has an average of about 1 toabout 8 drug molecules (e.g., maytansinoids) per antibody. In oneaspect, a composition comprising immunoconjugates has an average ofabout 2 to about 7 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 2 to about 6 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 2 to about 5 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 3 to about 5 drug molecules (e.g., maytansinoids) per antibody. Inone aspect, a composition comprising immunoconjugates has an average ofabout 3 to about 4 drug molecules (e.g., maytansinoids) per antibody.

In one aspect, a composition comprising immunoconjugates has an averageof about 2±0.5, about 3±0.5, about 4±0.5, about 5±0.5, about 6±0.5,about 7±0.5, or about 8±0.5 drug molecules (e.g., maytansinoids)attached per antibody. In one aspect, a composition comprisingimmunoconjugates has an average of about 3.5±0.5 drug molecules (e.g.,maytansinoids) per antibody.

The anti-CD37 antibody or fragment thereof can be modified by reacting abifunctional crosslinking reagent with the anti-CD37 antibody orfragment thereof, thereby resulting in the covalent attachment of alinker molecule to the anti-CD37 antibody or fragment thereof. As usedherein, a “bifunctional crosslinking reagent” is any chemical moietythat covalently links a cell-binding agent to a drug, such as the drugsdescribed herein. In another method, a portion of the linking moiety isprovided by the drug. In this respect, the drug comprises a linkingmoiety that is part of a larger linker molecule that is used to join thecell-binding agent to the drug. For example, to form the maytansinoidDM1, the side chain at the C-3 hydroxyl group of maytansine is modifiedto have a free sulfhydryl group (SH). This thiolated form of maytansinecan react with a modified cell-binding agent to form a conjugate.Therefore, the final linker is assembled from two components, one ofwhich is provided by the crosslinking reagent, while the other isprovided by the side chain from DM1.

The drug molecules can also be linked to the antibody molecules throughan intermediary carrier molecule such as serum albumin.

As used herein, the expression “linked to a cell-binding agent” or“linked to an anti-CD37 antibody or fragment” refers to the conjugatemolecule comprising at least one drug derivative bound to a cell-bindingagent anti-CD37 antibody or fragment via a suitable linking group, or aprecursor thereof. One linking group is SMCC.

In certain embodiments, cytotoxic agents useful in the present inventionare maytansinoids and maytansinoid analogs. Examples of suitablemaytansinoids include esters of maytansinol and maytansinol analogs.Included are any drugs that inhibit microtubule formation and that arehighly toxic to mammalian cells, as are maytansinol and maytansinolanalogs.

Examples of suitable maytansinol esters include those having a modifiedaromatic ring and those having modifications at other positions. Suchsuitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219;4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598;4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533;5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410;7,276,497 and 7,473,796.

In a certain embodiment, the immunoconjugates of the invention utilizethe thiol-containing maytansinoid (DM1), formally termedN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine, as thecytotoxic agent. DM1 is represented by the following structural formula(III):

In another embodiment, the conjugates of the present invention utilizethe thiol-containing maytansinoid N^(2′)-deacetyl-N^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) as thecytotoxic agent. DM4 is represented by the following structural formula(IV):

Another maytansinoid comprising a side chain that contains a stericallyhindered thiol bond isN^(2′)-deacetyl-N-^(2′)(4-mercapto-1-oxopentyl)-maytansine (termed DM3),represented by the following structural formula (V):

Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and7,276,497, can also be used in the conjugate of the present invention.In this regard, the entire disclosure of U.S. Pat. No. 5,208,020 andU.S. Pat. No. 7,276,697 is incorporated herein by reference.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. In some embodiments, the C-3 position servesas the position to chemically link the linking moiety, and in someparticular embodiments, the C-3 position of maytansinol serves as theposition to chemically link the linking moiety.

Structural representations of some conjugates are shown below:

Several descriptions for producing such antibody-maytansinoid conjugatesare provided in U.S. Pat. Nos. 6,333,410, 6,441,163, 6,716,821, and7,368,565, each of which is incorporated herein in its entirety.

In general, a solution of an antibody in aqueous buffer can be incubatedwith a molar excess of maytansinoids having a disulfide moiety thatbears a reactive group. The reaction mixture can be quenched by additionof excess amine (such as ethanolamine, taurine, etc.). Themaytansinoid-antibody conjugate can then be purified by gel filtration.

The number of maytansinoid molecules bound per antibody molecule can bedetermined by measuring spectrophotometrically the ratio of theabsorbance at 252 nm and 280 nm. The average number of maytansinoidmolecules/antibody can be, for example, about 1-10, 2-5, 3-4, or about3.5. In one aspect, the average number of maytansinoidmolecules/antibody is about 3.5±0.5.

Anthracycline compounds, as well as derivatives, intermediates andmodified versions thereof, can also be used to prepare anti-CD37immunoconjugates. For example, doxorubicin, doxorubicin derivatives,doxorubicin intermediates, and modified doxorubicins can be used inanti-CD37 conjugates. Exemplary compounds are described in WO2010/009124, which is herein incorporated by reference in its entirety.Such compounds include, for example, compounds of the following formula:

wherein R₁ is a hydrogen atom, hydroxy or methoxy group and R₂ is aC₁-C_(s) alkoxy group, or a pharmaceutically acceptable salt thereof.

Conjugates of antibodies with maytansinoid or other drugs can beevaluated for their ability to suppress proliferation of variousunwanted cell lines in vitro. For example, cell lines such as the humanlymphoma cell line Daudi and the human lymphoma cell line Ramos, caneasily be used for the assessment of cytotoxicity of these compounds.Cells to be evaluated can be exposed to the compounds for 4 to 5 daysand the surviving fractions of cells measured in direct assays by knownmethods. IC₅₀ values can then be calculated from the results of theassays.

The immunoconjugates can, according to some embodiments describedherein, be internalized into cells. The immunocongugate, therefore, canexert a therapeutic effect when it is taken up by, or internalized, by aCD37-expressing cell. In some particular embodiments, theimmunoconjugate comprises an antibody, antibody fragment, orpolypeptide, linked to a cytotoxic agent by a cleavable linker, and thecytotoxic agent is cleaved from the antibody, antibody fragment, orpolypeptide, wherein it is internalized by a CD37-expressing cell.

In some embodiments, the immunoconjugates are capable of reducing tumorvolume. For example, in some embodiments, treatment with animmunoconjugate results in a % T/C value that is less than about 50%,less than about 45%, less than about 40%, less than about 35%, less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 10%, or less than about 5%. In some particularembodiments, the immunoconjugates can reduce tumor size in a BJABxenograft model and/or a SU-DHL-4 xenograft model.

In another aspect of the invention siRNA molecules can be linked to theantibodies of the present invention instead of a drug. siRNAs can belinked to the antibodies of the present invention by methods commonlyused for the modification of oligonucleotides (see, for example, USPatent Publications 20050107325 and 20070213292). Thus the siRNA in its3′ or 5′-phosphoromidite form can be reacted with one end of thecrosslinker bearing a hydroxyl functionality to give an ester bondbetween the siRNA and the crosslinker. Similarly reaction of the siRNAphosphoramidite with a crosslinker bearing a terminal amino groupresults in linkage of the crosslinker to the siRNA through an amine.Alternatively, the siRNA can be derivatized by standard chemical methodsto introduce a thiol group. This thiol-containing siRNA can be reactedwith an antibody, that has been modified to introduce an activedisulfide or maleimide moiety, to produce a cleavable or non cleavableconjugate. Between 1-20 siRNA molecules can be linked to an antibody bythis method.

III. Polynucleotides

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide that specificallybinds CD37 or a fragment of such a polypeptide. For example, theinvention provides a polynucleotide comprising a nucleic acid sequencethat encodes an antibody to a human CD37 or encodes a fragment of suchan antibody. The polynucleotides of the invention can be in the form ofRNA or in the form of DNA. DNA includes cDNA, genomic DNA, and syntheticDNA; and can be double-stranded or single-stranded, and if singlestranded can be the coding strand or non-coding (anti-sense) strand.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotideencoding a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:4-120.

The invention further provides a polynucleotide comprising a sequenceselected from those shown in Tables 7-10 below.

TABLE 7 Variable heavy chain polynucleotide sequences AntibodyVH Polynucleotide Sequence (SEQ ID NO) muCD37-3caggtgcaggtgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactgtctcagggttctcattaaccacctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcacaaactatcattcagctctcaaatccagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagtctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctggtcacagtctctgca(SEQ ID NO: 121) chCD37-3aagcttgccaccatggctgtcctggcactgctcctctgcctggtgacatacccaagctgtgtcctatcacaggtgcaggtgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactgtctcagggttctcattaaccacctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcacaaactatcattcagctctcaaatccagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagtctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctggtcacagtctctgcagcctctacgaagggccc (SEQ ID NO: 122) huCD37-3v1.0aagcttgccaccatgggttggagctgcattattctgtttctggtggccaccgccaccggtgtgcactcacaagtccaagtccaagaatctggtccaggtctggtggccccttcccaaactctgagcatcacctgtaccgtttctggttttagccttaccacctctggtgtgagttgggtacgccaaccacccggtaagggtctcgaatggctgggtgtaatctggggtgatggttccacaaattaccatccttccctcaagtcccgccttagcatcaaaaaggatcacagcaaaagtcaagttttcctgaaactgaatagtctgacagcagccgatacagccacctactattgcgccaagggtggttatagtcttgcacactggggtcaaggtaccctcgttaccgtctcctcagctagtaccaagggccc (SEQ ID NO: 123) huCD37-3v1.1aagcttgccaccatgggctggagctgtatcattctgtttctggtggcgacagctactggggtccactcccaagtgcaggtacaagagtccgggcctggattggtcgcaccaagccagaccctctctatcacttgtaccgttagcgggttctctctgacaaccagtggagtgagttgggtgaggcagccaccaggaaagggactggagtggctgggggtgatttggggcgacggcagcacaaactatcattccagtcttaaatctcggttgtccattaaaaaagaccatagtaaatctcaagttttcctgaaactcaatagcctgacagccgcagacactgctacgtattactgcgccaaaggaggatacagtctggctcactggggacaggggaccctggtgaccgtgtcatccgcatcaacaaagggccc (SEQ ID NO: 124) muCD37-12cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaagtatggaatgaactgggtgaagcaggctcaaggaaagggtttaaagtggatgggctggataaacaccaacactggagagtcaagaaatgctgaagaattcaagggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaatatgaggacacggctacatatttctgtggaaggggcacggtagtagcggactggggccaaggcaccactctcacagtctcctca(SEQ ID NO: 125) chCD37-12aagcttgccaccatggggtggtcatgcataatcctctttctggtcgctactgctaccggtgtgcactcacagattcagctggttcaaagtggcccagagctgaaaaagccaggggaaacagtgaaaataagttgcaaggcatccggttacactttcacaaagtacggcatgaactgggtcaagcaggcccagggcaaggggctcaaatggatgggttggatcaataccaacactggcgagtctaggaatgctgaggagtttaagggccggtttgccttcagcctggagacaagtgccagcacagcttacctgcaaatcaacaatctgaagtatgaggatacagcaacctatttctgcggccgcggcactgtcgttgcagactggggacaaggtaccaccttgactgtatccagtgccagcactaagggccc (SEQ ID NO: 126) muCD37-38gatgtgcagcttcaggagtcaggacctgacctggtgaaaccttctcagtcactttcactcacctgcactgtcactggctactccatcaccagtggttttggctggcactggatccggcagtttccaggaaacaagctggaatggatggcctacatactctacagtggtggcactgactacaacccatctctcaaaagtcgaatctctatcactcgagacacttccaagaaccagttcttcctgcggttgagttctgtgactactgaggacacagccacatattactgtgcaagaggctactatggttacggggcctggtttgtttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 127) chCD37-38aagcttgccaccatgggctggagttgtatcattctgtttttggtggccaccgccactggagtccattcccaagtgcaactccaggaatctggccctgacctggttaagccatctcagagcctctccctgacctgcactgttacaggatactcaatcacatcaggctttggctggcactggatcagacaatttcccgggaacaagttggaatggatggcttacattctgtatagcgggggtaccgattacaatccttccctcaagagccgaatctctatcaccagggatacaagcaagaaccaattttttctccgcctcagctctgtgactaccgaagataccgctacttactattgtgccaggggctactatggatatggtgcatggttcgtctattggggccagggaaccctggtgactgtgagcgctgcctctaccaagggccc (SEQ ID NO: 128)huCD37-38aagcttgccaccatgggttggagctgcatcattcttttcctggtcgctactgcaactggagtccactcacaggtccagctgcaagagtccggtcctgggcttgtgaaacccagccagtccctcagtctcacctgtactgtctctggctactctattaccagtgggttcggctggcattggattaggcagtttcccggtaaggggctggagtggatggcatatatcctgtacagcggaggaaccgattacaacccaagtctgaagagcaggatcagcattacccgggacacaagcaaaaaccagtttttccttcggctgtctagtgttacagctgcagacaccgctacttactattgtgctcggggttactatggctatggggcttggtttgtgtattggggacaaggcactcttgtgaccgtgagcagcgcctcaacaaagggccc (SEQ ID NO: 129)muCD37-50gatgtgcagcttcaggagtcaggacctgacctgttgaaaccttctcagtcactttcactcacctgcactgtcactggctactccatcaccagtggttttgcctggcactggatccggcagtttccaggaaacaaactggaatggatgggctacatactctacagtggtagcactgtctacagcccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccacttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagagggtactatggttacggcgcctggtttgcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 130) huCD37-50aagcttgccaccatggggtggtcctgcataatccttttcctggttgctactgctaccggagtccattcacaggtgcagctgcaggagtccggccccggcctgctcaagccttctcagagtctgagtctgacttgtactgtttctggctacagcataaccagcggtttcgcttggcactggatcagacagcatcccggcaacaaactggagtggatgggatacatactgtactcaggctcaactgtctattccccctccctgaaatcccggatcagtattacccgtgacacttctaagaaccatttttttctgcagctgaacagcgttaccgcagctgacactgcaacctactactgtgcccggggatattatggatacggagcttggttcgcttactggggccaaggcaccctcgtaactgtgagtgctgcttccaccaagggccc (SEQ ID NO: 195)muCD37-51gatgtgcagcttcaggagtcaggacctgacctgttgaaaccttctcagtcactttcactcacctgcactgtcactggctactccatctccagtggttttgcctggcactggatccggcagtttccaggaaacaaactggaatggatgggctacatacactacagtggtagcactaactacagcccatctctcaaaagtcgaatctctatcactcgagactcatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagaggatactatggtttcggcgcctggtttgtttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 131) huCD37-51Aagcttgccaccatgggttggtcttgcatcatcctgttcctggtggccactgccactggcgtgcattcagaagttcagttggtggagtccggcccagaagtgctgaaacccggcgaatcactgtccctgacttgtaccgtgtcaggttatagcatcagcagcggctttgcttggcactggattcggcagtttccaggcaagggactggaatggatgggctacatccattacagtggctcaaccaattacagccctagcctgcagggccgaatctctattaccagggatagttctattaaccagtttttcctgcagcttaattccgtgactgcctctgacacagcaacttactattgcgcccgtggctactacgggttcggagcctggtttgtatactggggtcagggcaccctggtcactgtctcagccgcctctaccaagggccc (SEQ ID NO: 196)muCD37-56gatgtgcagcttcaggagtcaggacctgacctggtgaaaccttctcagtcactttcactcacctgcactgtcactggctactccatcaccagtggttttgcctggcactggatccggcagtttccaggaaacaaactggaatggatgggctacatacactacagtggtggcactaactacaacccatctctcaaaagtcgagtctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagaggctactatggtttcggggcctggtttgcttactggggccaagggactctggtccctgtctctgca (SEQ ID NO: 132) huCD37-56aagcttgccaccatggggtggagctgcattatcctgttcctcgtcgccaccgcaaccggcgtccactcccaggtgcagctgcaagaaagcgggccaggattggtaaaaccttcccagtctctgagtcttacttgtaccgtatctggatacagtatcacatctggcttcgcctggcattggattcgccagtttcccggcaaggggcttgagtggatggggtatattcattattctggaggtaccaactacaacccttccctgaagagtcgagtctcaattaccagggacacttccaagaaccaattctttttgcagcttaattcagtgaccgctgccgacaccgctacttactactgcgcccggggctactatgggtttggtgcctggttcgcctactggggccaggggaccctggtgcccgtgtctgctgcctccacaaagggccc (SEQ ID NO: 133)muCD37-57gatgtgcagcttcaggagtcaggacctgacctgttgaaaccttctcagtcactttcactcacctgcactgtcactggctactccatcaccagtggttttgcctggcactggatccggcagtttccaggaaacaaactggaatggatgggctacatactctacagtggtagcactgtctacagcccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagagggtactatggttacggcgcctggtttgcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 134) huCD37-57aagcttgccaccatgggctggagctgcatcattctgtttctggtggccacagcaactggcgttcacagtcaagtccaactgcaggagagcggccccggactcctgaaaccatctcagtcactcagtctgacatgtactgtgagcggctacagcattacctcaggcttcgcttggcattggatcaggcagttccccggaaaaggtctggagtggatggggtacattctgtacagcggcagtacagtgtattcaccctccttgaaatctaggatatcaatcacacgtgatacaagcaaaaatcagttcttcctccagctgaactccgtcaccgccgcagacacagcaacctattattgtgctcgcggatactacggatatggcgcatggttcgcctattggggccaggggacactcgtgaccgtttccgccgcctccacaaagggccc (SEQ ID NO: 135)

TABLE 8 Variable light chain polynucleotide sequences AntibodyVL Polynucleotide Sequence (SEQ ID NO) muCD37-3gacatccagatgactcagtctccagcctccctttctgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattcgcagtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcctggtcaatgttgcaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacagtattccctcaagatcaacagcctgcagtctgaagattttgggacttattactgtcaacattattggggtactacgtggacgttcggtggaggcaccaagctggaaatcaaacgt (SEQ ID NO: 136) chCD37-3gaattcgccaccatgagtgtgcccactcaggtcctggggttgctgctgctgtggcttacagatgccagatgtgacatccagatgactcagtctccagcctccctttctgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattcgcagtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcctggtcaatgttgcaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacagtattccctcaagatcaacagcctgcagtctgaagattttgggacttattactgtcaacattattggggtactacgtggacgttcggtggaggcaccaagctggaaatcaaacgtacg(SEQ ID NO: 137) huCD37-3gaattcgccaccatgggttggtcctgcatcatcttgtttctcgtggccacagccaccggtgttcactctgatatacaaat(1.0 and 1.1)gactcaaagcccttccagtttgagcgtaagtgtgggtgaacgcgtaacaatcacctgtagagctagtgaaaacatccgcagtaatctcgcatggtaccaacaaaagccaggtaagtcacctaagctcctcgtgaatgttgctaccaacctcgctgatggtgtgccttcacgattctctggttcaggttccggtaccgattattcacttaagatcaactcactccaaccagaagatttcggtacatattactgtcaacactactggggtacgacctggacattcggtcaaggtactaagctggaaatcaagcgtacg(SEQ ID NO: 138) muCD37-12gacattgtgctaacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagccaaagtgtcagtacatctagctatagttatttgtactggttccagcagaaaccaggacagccacccaaactcctcatcaagtatgcatccaacctagcatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatactgcaacatattactgtcaacacagttgggagattccgtacacgttcggaggggggaccaaactggaaataaaacgg(SEQ ID NO: 139) chCD37-12gaattcgccaccatgggttggtcctgtataatcctgttcttggtggccaccgctactggcgttcatagtgatattgtactcactcagtcaccagccagtctggcagtgtccctgggccagcgtgccaccatctcctgccgggcctcacagtccgtgagcactagctcttattcctatctctactggtttcaacagaagccaggacagccccctaagctgctgatcaagtacgcctccaacctcgccagcggcgttcccgctagattctctggttccggtagcggaactgatttcactttgaacatccaccccgttgaggaagaggataccgccacttactattgtcaacactcttgggagattccttacacctttggaggaggaacaaagctcgaaattaagcgtacg (SEQ ID NO: 140) muCD37-38caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcggtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggattagtaacccacccacgttcggaggggggaccaagctggaaattaaacgg (SEQ ID NO: 141) chCD37-38gaattcgccaccatgggctggtcctgtatcatcctgtttctcgtggccacagctacaggtgttcattctcagattgtgctgacccaatcaccagctattatgtccgctagccccggcgagaaagtgacaatgacatgtagcgctagctcttctgtgacttacatgcattggtatcaacagaagtcaggtaccagtcccaagcgttggatctacgacacatccaaactggcctccggagtccctgccaggttcagcggaggtgggtccggcaccagttattcactgaccatatcctctatggaagctgaagatgctgctacttattattgtcaacaatggatttctaacccccccacctttggtggcggaacaaagctggagatcaagcgtacg(SEQ ID NO: 142) huCD37-38gaattcgccaccatgggatggtcctgcattattctgttcttggtcgccactgctactggcgttcactctgacattgtgctcacacagtctccagcctcaatgtctgcttcccccggtgagcgggtgaccatgacatgctctgccagttcctccgtgacatatatgcattggtatcagcaaaaacccggtacctctccaaaaagatggatctacgacacttcaaagcttgcatcaggcgttcctgccagattttccgggtctgggtctggcacttcatacagtctgaccattagttccatggaagctgaagatgcagccacctattactgtcagcagtggatttcaaatcctcctaccttcggcggcggaaccaaactggagataaagcgtacg(SEQ ID NO: 143) muCD37-50caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccacctcaagtgtgacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactgccttatggagtccctggtcgtttcagtggtagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggagtgataacccacccacgttcggctcggggacaaagttggaaataaagcgg (SEQ ID NO: 144) huCD37-50gaattcgccaccatgggttggtcatgcattattctgttcctggttgctaccgcaacaggagtacatagtgagatagtcctcacccaaagtcctgctactatgtctgccagcccaggagagcgtgtgaccatgacttgctctgcaacctcaagtgtgacatacatgcattggtatcagcaaaagcctggccaatcccctaaaaggtggatctacgatacttctaatctgccatacggtgtgcccgcaaggttctccgggagtggcagtggcaccagttatagtctgaccatcagttcaatggaagcagaggatgcagcaacctattattgtcagcagtggtccgataatccccctacttttggtcagggtacaaagctggagattaagcgtacg(SEQ ID NO: 145) muCD37-51caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccacctcaagtgtgacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcaacatggaggctgaagatgctgccacttattactgccagcagtggagtagtaacccacccacgttcggctcggggacaaagttggaaataaagcgg (SEQ ID NO: 146) huCD37-51gaattcgccaccatgggatggagctgtattattctgttcctggttgctactgctactggcgtccattccgagatagtcctcacccagagccccgcaaccatgagtgcctcccctggggagcgagtgactatgacttgttccgccacttcttcagttacctatatgcattggtatcagcagaaacctggacagtctccaaagcgttggatttacgacacctccaacctggcttcaggagttcctgctaggttcagcggatctgggtctggcacaagttattcactcaccattagttccatggaggccgaagatgccgctacttactactgtcagcagtggagcagcaacccccctacattcgggcagggaactaagctggagatcaaacgtacg(SEQ ID NO: 147) muCD37-56caaattgttctcacccagtctccagcattcatgtctgcatctccaggggataaggtcaccatgacctgcagtgccagttcaagtgttacttacatgcactggtatcagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcggtgggtctgggacctcttactctctcacaatcagcaccatggaggctgaagatgctgccacttattactgccagcagtggattagtgacccacccacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 148) huCD37-56gaattcgccaccatgggctggtcctgtatcatcctgtttctggtggcaaccgctactggggttcactctgatattgtcctgacacagagtccagccttcatgagtgcttctcccggagaaaaggtcacaatgacttgttcagcttcctcctccgtcacatacatgcattggtaccagcagaagcctgaccagagtcctaagaggtggatctatgatacaagcaatctggcttccggtgtcccctcccgcttttcaggcggcggaagcggaactgactatagccttaccatctcctcaatggaagccgaggacgctgctacatattactgccagcaatggatcagcgaccctcctactttcggacagggaacaaaattggaaattaagcgtacg(SEQ ID NO: 149) muCD37-57caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccacctcaagtgtgacttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccagcagtggagtgataacccacccacgttcggctcggggacaaagttggaaataaagcgg (SEQ ID NO: 150) huCD37-57gaattcgccaccatggggtggtcctgtattatcctgttcctggtcgcaaccgccacaggcgttcactccgagatcgtgttgactcagagcccagccaccatgtccgcttcccccggggagagagtgacaatgacttgttccgccacaagttctgtaacctacatgcattggtaccagcaaaaaccaggacagagtccccgtcgttggatttatgatacctctaacctggcttcaggcgttcctgcccgcttttctggtagtggatctgggacttcctatagccttaccataagctctatggaagccgaggacgccgctacatactactgccagcagtggagtgataacccccccaccttcgggcagggaaccaaattggagatcaaacgtacg(SEQ ID NO: 151)

TABLE 9 Full-length heavy chain polynucleotide sequences AntibodyFull-Length Heavy Chain Polynucleotide Sequence (SEQ ID NO) chCD37-3aagcttgccaccatggctgtcctggcactgctcctctgcctggtgacatacccaagctgtgtcctatcacaggtgcaggtgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccattacatgcactgtctcagggttctcattaaccacctctggtgtaagctgggttcgccagcctccaggaaagggtctggagtggctgggagtaatatggggtgacgggagcacaaactatcattcagctctcaaatccagactgagcatcaagaaggatcactccaagagccaagttttcttaaaactgaacagtctgcaaactgatgacacagccacgtactactgtgccaaaggaggctactcgttggctcactggggccaagggactctggtcacagtctctgcagcctctacgaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 152)huCD37-3v1.0aagcttgccaccatgggttggagctgcattattctgtttctggtggccaccgccaccggtgtgcactcacaagtccaagtccaagaatctggtccaggtctggtggccccttcccaaactctgagcatcacctgtaccgtttctggttttagccttaccacctctggtgtgagttgggtacgccaaccacccggtaagggtctcgaatggctgggtgtaatctggggtgatggttccacaaattaccatccttccctcaagtcccgccttagcatcaaaaaggatcacagcaaaagtcaagttttcctgaaactgaatagtctgacagcagccgatacagccacctactattgcgccaagggtggttatagtcttgcacactggggtcaaggtaccctcgttaccgtctcctcagctagtaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 153)huCD37-3v1.1aagcttgccaccatgggctggagctgtatcattctgtttctggtggcgacagctactggggtccactcccaagtgcaggtacaagagtccgggcctggattggtcgcaccaagccagaccctctctatcacttgtaccgttagcgggttctctctgacaaccagtggagtgagttgggtgaggcagccaccaggaaagggactggagtggctgggggtgatttggggcgacggcagcacaaactatcattccagtcttaaatctcggttgtccattaaaaaagaccatagtaaatctcaagttttcctgaaactcaatagcctgacagccgcagacactgctacgtattactgcgccaaaggaggatacagtctggctcactggggacaggggaccctggtgaccgtgtcatccgcatcaacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 154)chCD37-12aagcttgccaccatggggtggtcatgcataatcctctttctggtcgctactgctaccggtgtgcactcacagattcagctggttcaaagtggcccagagctgaaaaagccaggggaaacagtgaaaataagttgcaaggcatccggttacactttcacaaagtacggcatgaactgggtcaagcaggcccagggcaaggggctcaaatggatgggttggatcaataccaacactggcgagtctaggaatgctgaggagtttaagggccggtttgccttcagcctggagacaagtgccagcacagcttacctgcaaatcaacaatctgaagtatgaggatacagcaacctatttctgcggccgcggcactgtcgttgcagactggggacaaggtaccaccttgactgtatccagtgccagcactaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag (SEQ ID NO: 155)chCD37-38aagcttgccaccatgggctggagttgtatcattctgtttttggtggccaccgccactggagtccattcccaagtgcaactccaggaatctggccctgacctggttaagccatctcagagcctctccctgacctgcactgttacaggatactcaatcacatcaggctttggctggcactggatcagacaatttcccgggaacaagttggaatggatggcttacattctgtatagcgggggtaccgattacaatccttccctcaagagccgaatctctatcaccagggatacaagcaagaaccaattttttctccgcctcagctctgtgactaccgaagataccgctacttactattgtgccaggggctactatggatatggtgcatggttcgtctattggggccagggaaccctggtgactgtgagcgctgcctctaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 156) huCD37-38aagcttgccaccatgggttggagctgcatcattcttttcctggtcgctactgcaactggagtccactcacaggtccagctgcaagagtccggtcctgggcttgtgaaacccagccagtccctcagtctcacctgtactgtctctggctactctattaccagtgggttcggctggcattggattaggcagtttcccggtaaggggctggagtggatggcatatatcctgtacagcggaggaaccgattacaacccaagtctgaagagcaggatcagcattacccgggacacaagcaaaaaccagtttttccttcggctgtctagtgttacagctgcagacaccgctacttactattgtgctcggggttactatggctatggggcttggtttgtgtattggggacaaggcactcttgtgaccgtgagcagcgcctcaacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 157) huCD37-50aagcttgccaccatggggtggtcctgcataatccttttcctggttgctactgctaccggagtccattcacaggtgcagctgcaggagtccggccccggcctgctcaagccttctcagagtctgagtctgacttgtactgtttctggctacagcataaccagcggtttcgcttggcactggatcagacagcatcccggcaacaaactggagtggatgggatacatactgtactcaggctcaactgtctattccccctccctgaaatcccggatcagtattacccgtgacacttctaagaaccatttttttctgcagctgaacagcgttaccgcagctgacactgcaacctactactgtgcccggggatattatggatacggagcttggttcgcttactggggccaaggcaccctcgtaactgtgagtgctgcttccaccaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 158) huCD37-51aagcttgccaccatgggttggtcttgcatcatcctgttcctggtggccactgccactggcgtgcattcagaagttcagttggtggagtccggcccagaagtgctgaaacccggcgaatcactgtccctgacttgtaccgtgtcaggttatagcatcagcagcggctttgcttggcactggattcggcagtttccaggcaagggactggaatggatgggctacatccattacagtggctcaaccaattacagccctagcctgcagggccgaatctctattaccagggatagttctattaaccagtttttcctgcagcttaattccgtgactgcctctgacacagcaacttactattgcgcccgtggctactacgggttcggagcctggtttgtatactggggtcagggcaccctggtcactgtctcagccgcctctaccaagggcccatcagttttcccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgagttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 159) huCD37-56aagcttgccaccatggggtggagctgcattatcctgttcctcgtcgccaccgcaaccggcgtccactcccaggtgcagctgcaagaaagcgggccaggattggtaaaaccttcccagtctctgagtcttacttgtaccgtatctggatacagtatcacatctggcttcgcctggcattggattcgccagtttcccggcaaggggcttgagtggatggggtatattcattattctggaggtaccaactacaacccttccctgaagagtcgagtctcaattaccagggacacttccaagaaccaattctttttgcagcttaattcagtgaccgctgccgacaccgctacttactactgcgcccggggctactatgggtttggtgcctggttcgcctactggggccaggggaccctggtgcccgtgtctgctgcctccacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 160) huCD37-57aagcttgccaccatgggctggagctgcatcattctgtttctggtggccacagcaactggcgttcacagtcaagtccaactgcaggagagcggccccggactcctgaaaccatctcagtcactcagtctgacatgtactgtgagcggctacagcattacctcaggcttcgcttggcattggatcaggcagttccccggaaaaggtctggagtggatggggtacattctgtacagcggcagtacagtgtattcaccctccttgaaatctaggatatcaatcacacgtgatacaagcaaaaatcagttcttcctccagctgaactccgtcaccgccgcagacacagcaacctattattgtgctcgcggatactacggatatggcgcatggttcgcctattggggccaggggacactcgtgaccgtttccgccgcctccacaaagggcccatcagttttccccttggctccaagttctaaatccacaagcggtggaacagctgcactgggatgcctcgttaaagattatttccctgagcctgtgacagtgagctggaatagcggagcattgacttcaggtgtgcacacttttcccgctgtgttgcagtcctccggtctgtactcactgtccagtgtcgtaaccgtcccttctagcagcttgggaacccagacctacatctgtaacgtcaaccataaaccatccaacacaaaggtggataagaaggttgaaccaaagagctgtgataagacacatacatgccctccttgtcctgcaccagagctcctcggaggtccatctgtgttcctgtttccccccaaacccaaggacactcttatgatctctcgtactccagaggtcacctgtgttgttgtcgacgtgagccatgaagatcccgaggttaaattcaactggtacgtggatggagtcgaggttcacaatgccaagaccaagcccagggaggagcaatataattctacatatcgggtagtgagcgttctgaccgtgctccaccaagattggctcaatggaaaagagtacaagtgcaaggtgtccaacaaggctcttcccgctcccattgagaaaactatctccaaagccaaggggcagccacgggaaccccaggtgtatacattgcccccatctagagacgagctgaccaagaaccaggtgagtctcacttgtctggtcaaggggttttacccttctgacattgctgtagagtgggagtctaacggacagccagaaaacaactacaagacaactcccccagtgctggacagcgacgggagcttcttcctctactccaagttgactgtagacaagtctagatggcagcaaggaaacgttttctcctgctcagtaatgcatgaggctctgcacaatcactatacccagaaatcactgtcccttagcccagggtgactcgag(SEQ ID NO: 161)

TABLE 10 Full-length light chain polynucleotide sequences AntibodyFull-length Light Chain Polynucleotide Sequence (SEQ ID NO) chCD37-3gaattcgccaccatgagtgtgcccactcaggtcctggggttgctgctgctgtggcttacagatgccagatgtgacatccagatgactcagtctccagcctccctttctgtatctgtgggagaaactgtcaccatcacatgtcgagcaagtgagaatattcgcagtaatttagcatggtatcagcagaaacagggaaaatctcctcagctcctggtcaatgttgcaacaaacttagcagatggtgtgccatcaaggttcagtggcagtggatcaggcacacagtattccctcaagatcaacagcctgcagtctgaagattttgggacttattactgtcaacattattggggtactacgtggacgttcggtggaggcaccaagctggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 162) huCD37-3gaattcgccaccatgggttggtcctgcatcatcttgtttctcgtggccacagccaccggtgttcactctgatatacaaat(1.0 and 1.1)gactcaaagcccttccagtttgagcgtaagtgtgggtgaacgcgtaacaatcacctgtagagctagtgaaaacatccgcagtaatctcgcatggtaccaacaaaagccaggtaagtcacctaagctcctcgtgaatgttgctaccaacctcgctgatggtgtgccttcacgattctctggttcaggttccggtaccgattattcacttaagatcaactcactccaaccagaagatttcggtacatattactgtcaacactactggggtacgacctggacattcggtcaaggtactaagctggaaatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 163) chCD37-12gaattcgccaccatgggttggtcctgtataatcctgttcttggtggccaccgctactggcgttcatagtgatattgtactcactcagtcaccagccagtctggcagtgtccctgggccagcgtgccaccatctcctgccgggcctcacagtccgtgagcactagctcttattcctatctctactggtttcaacagaagccaggacagccccctaagctgctgatcaagtacgcctccaacctcgccagcggcgttcccgctagattctctggttccggtagcggaactgatttcactttgaacatccaccccgttgaggaagaggataccgccacttactattgtcaacactcttgggagattccttacacctttggaggaggaacaaagctcgaaattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 164) chCD37-38gaattcgccaccatgggctggtcctgtatcatcctgtttctcgtggccacagctacaggtgttcattctcagattgtgctgacccaatcaccagctattatgtccgctagccccggcgagaaagtgacaatgacatgtagcgctagctcttctgtgacttacatgcattggtatcaacagaagtcaggtaccagtcccaagcgttggatctacgacacatccaaactggcctccggagtccctgccaggttcagcggaggtgggtccggcaccagttattcactgaccatatcctctatggaagctgaagatgctgctacttattattgtcaacaatggatttctaacccccccacctttggtggcggaacaaagctggagatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 165) huCD37-38gaattcgccaccatgggatggtcctgcattattctgttcttggtcgccactgctactggcgttcactctgacattgtgctcacacagtctccagcctcaatgtctgcttcccccggtgagcgggtgaccatgacatgctctgccagttcctccgtgacatatatgcattggtatcagcaaaaacccggtacctctccaaaaagatggatctacgacacttcaaagcttgcatcaggcgttcctgccagattttccgggtctgggtctggcacttcatacagtctgaccattagttccatggaagctgaagatgcagccacctattactgtcagcagtggatttcaaatcctcctaccttcggcggcggaaccaaactggagataaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 166) huCD37-50gaattcgccaccatgggttggtcatgcattattctgttcctggttgctaccgcaacaggagtacatagtgagatagtcctcacccaaagtcctgctactatgtctgccagcccaggagagcgtgtgaccatgacttgctctgcaacctcaagtgtgacatacatgcattggtatcagcaaaagcctggccaatcccctaaaaggtggatctacgatacttctaatctgccatacggtgtgcccgcaaggttctccgggagtggcagtggcaccagttatagtctgaccatcagttcaatggaagcagaggatgcagcaacctattattgtcagcagtggtccgataatccccctacttttggtcagggtacaaagctggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 167) huCD37-51gaattcgccaccatgggttggtcatgcattattctgttcctggttgctaccgcaacaggagtacatagtgagatagtcctcacccagagccccgcaaccatgagtgcctcccctggggagcgagtgactatgacttgttccgccacttcttcagttacctatatgcattggtatcagcagaaacctggacagtctccaaagcgttggatttacgacacctccaacctggcttcaggagttcctgctaggttcagcggatctgggtctggcacaagttattcactcaccattagttccatggaggccgaagatgccgctacttactactgtcagcagtggagcagcaacccccctacattcgggcagggaactaagctggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 168) huCD37-56gaattcgccaccatgggctggtcctgtatcatcctgtttctggtggcaaccgctactggggttcactctgatattgtcctgacacagagtccagccttcatgagtgcttctcccggagaaaaggtcacaatgacttgttcagcttcctcctccgtcacatacatgcattggtaccagcagaagcctgaccagagtcctaagaggtggatctatgatacaagcaatctggcttccggtgtcccctcccgcttttcaggcggcggaagcggaactgactatagccttaccatctcctcaatggaagccgaggacgctgctacatattactgccagcaatggatcagcgaccctcctactttcggacagggaacaaaattggaaattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 169) huCD37-57gaattcgccaccatggggtggtcctgtattatcctgttcctggtcgcaaccgccacaggcgttcactccgagatcgtgttgactcagagcccagccaccatgtccgcttcccccggggagagagtgacaatgacttgttccgccacaagttctgtaacctacatgcattggtaccagcaaaaaccaggacagagtccccgtcgttggatttatgatacctctaacctggcttcaggcgttcctgcccgcttttctggtagtggatctgggacttcctatagccttaccataagctctatggaagccgaggacgccgctacatactactgccagcagtggagtgataacccccccaccttcgggcagggaaccaaattggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 170)

Also provided is a polynucleotide having at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to SEQ ID NOs:121-170. Thus, in certain embodiments,the polypeptide comprises (a) a polypeptide having at least about 95%sequence identity to SEQ ID NOs:121-135 or 152-161, and/or (b) apolypeptide having at least about 95% sequence identity to SEQ IDNOs:136-151 or 162-170. In certain embodiments, the polypeptidecomprises (a) a polypeptide having the amino acid sequence of SEQ IDNOs: 121-135 or 152-161; and/or (b) a polypeptide having the amino acidsequence of SEQ ID NOs: 136-151 or 162-170.

In some embodiments, the polynucleotide encodes the light chain encodedby the recombinant plasmid DNA phuCD37-3LC (ATCC Deposit DesignationPTA-10722, deposited with the ATCC on Mar. 18, 2010) or a light chainthat is at least about 85%, at least about 90%, at least about 95%, orat least about 99% to the light chain encoded by phuCD37-3LC(PTA-10722). In some embodiments, the polynucleotide encodes the heavychain encoded by the recombinant plasmid DNA phuCD37-3HCv.1.0 (ATCCDeposit Designation PTA-10723, deposited with the ATCC on Mar. 18, 2010)or a heavy chain that is at least about 85%, at least about 90%, atleast about 95%, or at least about 99% identical to the heavy chainencoded by phuCD37-3HCv.1.0 (PTA-10723). In certain embodiments thepolynucleotide is the recombinant plasmid DNA phuCD37-3LC (PTA-10722) orthe recombinant plasmid phuCD37-3HCv.1.0 (PTA-10723).

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g. a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells)is used.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Vectors and cells comprising the polynucleotides described herein arealso provided.

IV. Methods of Use and Pharmaceutical Compositions

The CD37-binding agents (including antibodies, immunoconjugates, andpolypeptides) of the invention are useful in a variety of applicationsincluding, but not limited to, therapeutic treatment methods, such asthe treatment of cancer, such as B-cell malignancies. In certainembodiments, the agents are useful for inhibiting tumor growth, inducingdifferentiation, reducing tumor volume, and/or reducing thetumorigenicity of a tumor. The methods of use can be in vitro, ex vivo,or in vivo methods. In certain embodiments, the CD37-binding agent orantibody or immunoconjugate, or polypeptide is an antagonist of thehuman CD37 to which it binds.

In one aspect, anti-CD37 antibodies and immunoconjugates of theinvention are useful for detecting the presence of CD37 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, such tissues includenormal and/or cancerous tissues that express CD37 at higher levelsrelative to other tissues, for example, B cells and/or B cell associatedtissues.

In one aspect, the invention provides a method of detecting the presenceof CD37 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-CD37 antibodyunder conditions permissive for binding of the anti-CD37 antibody toCD37, and detecting whether a complex is formed between the anti-CD37antibody and CD37.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of CD37. In certain embodiments,the method comprises contacting a test cell with an anti-CD37 antibody;determining the level of expression (either quantitatively orqualitatively) of CD37 by the test cell by detecting binding of theanti-CD37 antibody to CD37; and comparing the level of expression ofCD37 by the test cell with the level of expression of CD37 by a controlcell (e.g., a normal cell of the same tissue origin as the test cell ora cell that expresses CD37 at levels comparable to such a normal cell),wherein a higher level of expression of CD37 by the test cell ascompared to the control cell indicates the presence of a disorderassociated with increased expression of CD37. In certain embodiments,the test cell is obtained from an individual suspected of having adisorder associated with increased expression of CD37. In certainembodiments, the disorder is a cell proliferative disorder, such as acancer or a tumor.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-CD37antibody to CD37 expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing CD37 on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-CD37 antibody under conditions permissive for binding of theanti-CD37 antibody to CD37, and detecting whether a complex is formedbetween the anti-CD37 antibody and CD37 on the cell surface. Anexemplary assay for detecting binding of an anti-CD37 antibody to CD37expressed on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-CD37antibodies to CD37. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-CD37 antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction.

In certain embodiments, anti-CD37 antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-CD37antibody from any CD37 that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-CD37antibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-CD37 antibody afterformation of a complex between the anti-CD37 antibody and CD37, e.g., byimmunoprecipitation.

Any of the above embodiments of diagnosis or detection can be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-CD37 antibody.

In certain embodiments, the disease treated with the CD37-binding agentor antagonist (e.g., an anti-CD37 antibody) is a cancer. In certainembodiments, the cancer is characterized by CD37 expressing cells towhich the CD37-binding agent (e.g., antibody) binds.

The present invention provides for methods of treating cancer comprisingadministering a therapeutically effective amount of a CD37-binding agentto a subject (e.g., a subject in need of treatment). In certainembodiments, the cancer is a B-cell malignancy. In certain embodiments,the cancer is selected from the group consisting of B cell lymphomas,NHL, precursor B cell lymphoblastic leukemia/lymphoma and mature B cellneoplasms, B cell chronic lymphocytic leukemia (CLL)/small lymphocyticlymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacyticlymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), lowgrade, intermediate-grade and high-grade (FL), cutaneous follicle centerlymphoma, marginal zone B cell lymphoma, MALT type marginal zone B celllymphoma, nodal marginal zone B cell lymphoma, splenic type marginalzone B cell lymphoma, hairy cell leukemia, diffuse large B celllymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma,post-transplant lymphoproliferative disorder, Waldenstrom'smacroglobulinemia, and anaplastic large-cell lymphoma (ALCL). In certainembodiments, the subject is a human.

The present invention further provides methods for inhibiting tumorgrowth using the antibodies or other agents described herein. In certainembodiments, the method of inhibiting the tumor growth comprisescontacting the cell with a CD37-binding agent (e.g., antibody) in vitro.For example, an immortalized cell line or a cancer cell line thatexpresses CD37 is cultured in medium to which is added the antibody orother agent to inhibit tumor growth. In some embodiments, tumor cellsare isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and cultured in medium towhich is added an CD37-binding agent to inhibit tumor growth.

In some embodiments, the method of inhibiting tumor growth comprisescontacting the tumor or tumor cells with the CD37-binding agent (e.g.,antibody) in vivo. In certain embodiments, contacting a tumor or tumorcell with a CD37-binding agent is undertaken in an animal model. Forexample, CD37-binding agents can be administered to xenograftsexpressing one or more CD37s that have been grown in immunocompromisedmice (e.g. NOD/SCID mice) to inhibit tumor growth. In some embodiments,cancer stem cells are isolated from a patient sample such as, forexample, a tissue biopsy, pleural effusion, or blood sample and injectedinto immunocompromised mice that are then administered a CD37-bindingagent to inhibit tumor cell growth. In some embodiments, theCD37-binding agent is administered at the same time or shortly afterintroduction of tumorigenic cells into the animal to prevent tumorgrowth. In some embodiments, the CD37-binding agent is administered as atherapeutic after the tumorigenic cells have grown to a specified size.

In certain embodiments, the method of inhibiting tumor growth comprisesadministering to a subject a therapeutically effective amount of aCD37-binding agent. In certain embodiments, the subject is a human. Incertain embodiments, the subject has a tumor or has had a tumor removed.

In certain embodiments, the tumor expresses the CD37 to which theCD37-binding agent or antibody binds. In certain embodiments, the tumoroverexpresses the human CD37.

In addition, the invention provides a method of reducing thetumorigenicity of a tumor in a subject, comprising administering atherapeutically effective amount of a CD37-binding agent to the subject.In certain embodiments, the tumor comprises cancer stem cells. Incertain embodiments, the frequency of cancer stem cells in the tumor isreduced by administration of the agent.

The invention further provides methods of differentiating tumorigeniccells into non-tumorigenic cells comprising contacting the tumorigeniccells with a CD37-binding agent (for example, by administering theCD37-binding agent to a subject that has a tumor comprising thetumorigenic cells or that has had such a tumor removed.

The use of the CD37-binding agents, polypeptides, or antibodiesdescribed herein to induce the differentiation of cells, including, butnot limited to tumor cells, is also provided. For example, methods ofinducing cells to differentiate comprising contacting the cells with aneffective amount of a CD37-binding agent (e.g., an anti-CD37 antibody)described herein are envisioned. Methods of inducing cells in a tumor ina subject to differentiate comprising administering a therapeuticallyeffective amount of a CD37-binding agent, polypeptide, or antibody tothe subject are also provided. In certain embodiments, the tumor is apancreatic tumor. In certain other embodiments, the tumor is a colontumor. In some embodiments, the treatment methods comprise administeringa therapeutically effective amount of the CD37-binding agent,polypeptide, or antibody to the subject.

The present invention further provides pharmaceutical compositionscomprising one or more of the CD37-binding agents described herein. Incertain embodiments, the pharmaceutical compositions further comprise apharmaceutically acceptable vehicle. These pharmaceutical compositionsfind use in inhibiting tumor growth and treating cancer in humanpatients.

In certain embodiments, formulations are prepared for storage and use bycombining a purified antibody or agent of the present invention with apharmaceutically acceptable vehicle (e.g. carrier, excipient)(Remington, The Science and Practice of Pharmacy 20th Edition MackPublishing, 2000). Suitable pharmaceutically acceptable vehiclesinclude, but are not limited to, nontoxic buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (e.g.octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight polypeptides (e.g. less than about 10 amino acid residues);proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, histidine, arginine, or lysine; carbohydratessuch as monosacchandes, disaccharides, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g. Zn-protein complexes); and non-ionic surfactants such asTWEEN or polyethylene glycol (PEG).

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

An antibody or immunoconjugate of the invention can be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-cancer properties. Thesecond compound of the pharmaceutical combination formulation or dosingregimen can have complementary activities to the ADC of the combinationsuch that they do not adversely affect each other. Pharmaceuticalcompositions comprising the CD37-binding agent and the secondanti-cancer agent are also provided. For example, CD37-binding agentscan be administered in combination with CD20 antagonists, such asRituximab.

For the treatment of the disease, the appropriate dosage of an antibodyor agent of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antibody or agent is administered fortherapeutic or preventative purposes, previous therapy, patient'sclinical history, and so on all at the discretion of the treatingphysician. The antibody or agent can be administered one time or over aseries of treatments lasting from several days to several months, oruntil a cure is effected or a diminution of the disease state isachieved (e.g. reduction in tumor size). Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient and will vary depending on the relative potency of an individualantibody or agent. The administering physician can easily determineoptimum dosages, dosing methodologies and repetition rates. In certainembodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, andcan be given once or more daily, weekly, monthly or yearly. In certainembodiments, the antibody or other CD37-binding agent is given onceevery two weeks or once every three weeks. In certain embodiments, thedosage of the antibody or other CD37-binding agent is from about 0.1 mgto about 20 mg per kg of body weight. The treating physician canestimate repetition rates for dosing based on measured residence timesand concentrations of the drug in bodily fluids or tissues.

The combination therapy can provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

VI. Kits Comprising CD37 Binding Agents

The present invention provides kits that comprise the antibodies,immunoconjugates or other agents described herein and that can be usedto perform the methods described herein. In certain embodiments, a kitcomprises at least one purified antibody against CD37 in one or morecontainers. In some embodiments, the kits contain all of the componentsnecessary and/or sufficient to perform a detection assay, including allcontrols, directions for performing assays, and any necessary softwarefor analysis and presentation of results. One skilled in the art willreadily recognize that the disclosed antibodies, immunoconjugates orother agents of the present invention can be readily incorporated intoone of the established kit formats which are well known in the art.

Further provided are kits comprising a CD37-binding agent (e.g., aCD37-binding antibody), as well as a second anti-cancer agent. Incertain embodiments, the second anti-cancer agent is a chemotherapeuticagent (e.g., rituximab).

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe indetail preparation of certain antibodies of the present disclosure andmethods for using antibodies of the present disclosure. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, can be practiced without departing from the scopeof the present disclosure.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application

Cell Lines and Growth

Cell line Origin Source Ramos Burkitt lymphoma DSMZ (ACC 603) RajiBurkitt lymphoma DSMZ (ACC 319) Daudi Burkitt lymphoma DSMZ (ACC 78)Namalwa Burkitt lymphoma ATCC (CRL-1432) BJAB B-NHL A gift from ElliotKieff (Harvard) WSU- B-NHL, diffuse large B-cell lymphoma DSMZ (ACC 575)DLCL-2 RL B-NHL, diffuse large B-cell lymphoma DSMZ (ACC 613) SU- B-NHL,diffuse histiocytic lymphoma DSMZ (ACC 495) DHL-4 DOHH-2 refractoryimmunoblastic B cell DSMZ (ACC 47) lymphoma, follicular lymphoma Granta-B-NHL, mantle cell lymphoma DSMZ (ACC 342) 519

All cell lines were grown in RPMI-1640 media supplemented with 10% fetalbovine serum, 2 mM glutamine and 1% penicillin-streptomycin (allreagents from Invitrogen) at 37° C. in a humidified 5% CO₂ incubator.Cells were passaged by diluting into fresh media twice per week andmaintained between 0.2 to 1×10⁶ cells/ml.

Example 1 Production of Murine CD37 Antibodies

An expression plasmid pSRa-CD37 was constructed that contained theentire CD37 coding sequence (CDS) flanked by XbaI and BamHI restrictionsites that allowed expression of human CD37. 300-19 cells, a pre-B cellline derived from a Balb/c mouse (M. G. Reth et al. 1985, Nature, 317:353-355), were transfected with this expression plasmid to stablyexpress high levels of human CD37 on the cell surface and used forimmunization of Balb/c VAF mice. Mice were subcutaneously immunized withapproximately 5×10⁶ CD37-expressing 300-19 cells per mouse every 2-3weeks by standard immunization protocols used at ImmunoGen, Inc. Theimmunized mice were boosted with another dose of antigen three daysbefore being sacrificed for hybridoma generation. The spleen from themouse was collected according to standard animal protocols and wasground between two sterile, frosted microscopic slides to obtain asingle cell suspension in RPMI-1640 medium. The spleen cells werepelleted, washed, and fused with murine myeloma P3X63Ag8.653 cells (J.F. Kearney et al. 1979, J Immunol, 123: 1548-1550) by using polyethyleneglycol-1500 (Roche 783 641). The fused cells were resuspended inRPMI-1640 selection medium containing hypoxanthine-aminopterin-thymidine(HAT) (Sigma H-0262) and selected for growth in 96-well flat-bottomedculture plates (Corning-Costar 3596, 200 μL of cell suspension per well)at 37° C. with 5% CO₂. After 5 days of incubation, 100 μL of culturesupernatant were removed from each well and replaced with 100 μL ofRPMI-1640 medium containing hypoxanthine-thymidine (HT) supplement(Sigma H-0137). Incubation at 37° C. with 5% CO₂ was continued untilhydridoma clones were ready for antibody screening. Other techniques ofimmunization and hybridoma production can also be used, including thosedescribed in J. Langone and H. Vunakis (Eds., Methods in Enzymology,Vol. 121, “Immunochemical Techniques, Part I”; Academic Press, Florida)and E. Harlow and D. Lane (“Antibodies: A Laboratory Manual”; 1988; ColdSpring Harbor Laboratory Press, New York).

Hybridoma Screening and Selection

Culture supernatants from the hybridoma were screened by flow cytometryfor secretion of mouse monoclonal antibodies that bind to theCD37-expressing 300-19 cells, but not to the non-transfected 300-19cells. 100 μl of hybridoma supernatants was incubated for 3 h witheither CD37-expressing 300-19 cells or the non-transfected 300-19 cells(1×10⁵ cells per sample) in 100 μL FACS buffer (RPMI-1640 mediumsupplemented with 2% normal goat serum). Then, the cells were pelleted,washed, and incubated for 1 h with 100 μL of PE-conjugated goatanti-mouse IgG-antibody (Jackson Laboratory, 6 μg/mL in FACS buffer).The cells were pelleted again, washed with FACS buffer and resuspendedin 200 μL of PBS containing 1% formaldehyde. Samples were acquired usinga FACSCalibur flow cytometer with the HTS multiwell sampler or a FACSarray flow cytometer and analyzed using CellQuest Pro (all from BDBiosciences, San Diego, US).

The hybridoma clones that tested positive were subcloned by limitingdilution. One subclone from each hybridoma, which showed the samereactivity against CD37 as the parental cells by flow cytometry, waschosen for subsequent analysis. Stable subclones were cultured and theisotype of each secreted anti-CD37 antibody was identified usingcommercial isotyping reagents (Roche 1493027).

A total of 45 separate fusion experiments were conducted over the courseof this investigation. A single fusion experiment routinely yieldedapproximately between 200 and 1000 hybridoma clones. All the resultinghybridoma clones were screened for CD37 binding by flow cytometry and atotal of 184 hybridoma clones showed specific binding to CD37.

Antibody Purification

Antibodies were purified from hybridoma subclone supernatants usingstandard methods, such as, for example Protein A or G chromatography(HiTrap Protein A or G HP, 1 mL, Amersham Biosciences). Briefly,supernatant was prepared for chromatography by the addition of 1/10volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjusted supernatant wasfiltered through a 0.22 μm filter membrane and loaded onto columnequilibrated with binding buffer (PBS, pH 7.3). The column was washedwith binding buffer until a stable baseline was obtained with noabsorbance at 280 nm. Antibody was eluted with 0.1 M acetic acid buffercontaining 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 mL/min.Fractions of approximately 0.25 mL were collected and neutralized by theaddition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) wasdialyzed overnight twice against 1×PBS and sterilized by filteringthrough a 0.2 μm filter membrane. Purified antibody was quantified byabsorbance at A280.

Protein A purified fractions were further polished using ion exchangechromatography (IEX) with quaternary ammonium (Q) chromatography formurine antibodies. Briefly, samples from protein A purification werebuffer exchanged into binding buffer (10 mM Tris, 10 mM sodium chloride,pH 8.0) and filtered through 0.22 μm filer. The prepared sample was thenloaded onto a Q fast flow resin (GE Lifesciences) that was equilibratedwith binding buffer at a flow rate of 120 cm/hr. Column size was chosento have sufficient capacity to bind all the MAb in the sample. Thecolumn was then washed with binding buffer until a stable baseline wasobtained with no absorbance at 280 nm. Antibody was eluted by initiatinga gradient from 10 mM to 500 mM sodium chloride in 20 column volume(CV). Peak fractions were collected based on absorbance measurement at280 nm (A280). The percentage of monomer was assessed with sizeexclusion chromatography (SEC) on a TSK gel G3000SWXL, 7.8×300 mm with aSWXL guard column, 6.0×40 mm (Tosoh Bioscience, Montgomeryville, Pa.)using an Agilent HPLC 1100 system (Agilent, Santa Clara, Calif.).Fractions with monomer content above 95% were pooled, buffer exchangedto PBS (pH 7.4) using a TFF system, and sterilized by filtering througha 0.2 μm filter membrane. The IgG concentration of purified antibody wasdetermined by A280 using an extinction coefficient of 1.47. Alternativemethods such as ceramic hydroxyapatite (CHT) were also used to polishantibodies with good selectivity. Type II CHT resin with 40 μm particlesize (Bio-Rad Laboratories) were used with a similar protocol asdescribed for IEX chromatography. The binding buffer for CHT correspondsto 20 mM sodium phosphate, pH 7.0 and antibody was eluted with agradient of 20-160 mM sodium phosphate over 20 CV.

Example 2 Binding Characterization by Flow Cytometry

Binding specificity was tested by flow cytometry using purifiedantibodies. FACS histograms demonstrating the binding of muCD37-3,muCD37-12, muCD37-38, muCD37-50, muCD37-51, muCD37-56 and muCD37-57 toCD37-expressing 300-19 cells and the absence of binding to the parental300-19 cells are shown in FIG. 1 and FIG. 2. All murine antibodies wereincubated for 3 h with either CD37-expressing 300-19 cells or thenon-transfected 300-19 cells (1×10⁵ cells per sample) in 100 μL FACSbuffer (RPMI-1640 medium supplemented with 2% normal goat serum). Then,the cells were pelleted, washed, and incubated for 1 h with 100 μL ofFITC-conjugated goat anti-mouse IgG-antibody (Jackson Laboratory, 6μg/mL in FACS buffer). The cells were pelleted again, washed with FACSbuffer and resuspended in 200 μL of PBS containing 1% formaldehyde.Samples were acquired using a FACSCalibur flow cytometer with the HTSmultiwell sampler or a FACS array flow cytometer and analyzed usingCellQuest Pro (all from BD Biosciences, San Diego, US).

The FACS histograms of CD37-expressing 300-19 cells incubated withmuCD37-3, muCD37-12, muCD37-38, muCD37-50, muCD37-51, muCD37-56 ormuCD37-57 showed a fluorescence shift, while parental 300-19 cells didnot. Also, no significant fluorescence shift was detected when eithercell lines was incubated only with FITC-conjugated goat anti-mouseIgG-antibody alone (FIG. 1 bottom).

To verify that the antibodies can also bind to endogenously expressedCD37, binding experiments were performed with CD37-positive WSU-DLCL-2lymphoma cells and the muCD37-3, muCD37-12, muCD37-8, muCD37-10 ormuCD37-14 antibodies. WSU-DLCL-2 cells were incubated with varyingconcentrations of murine antibodies and processed as described above forflow cytometry analysis. Data analysis was performed using CellQuest Pro(BD Biosciences, San Diego, US) and for each sample the meanfluorescence intensity for FL1 (MFI) was exported and plotted againstthe antibody concentration in a semi-log plot (FIG. 3). A dose-responsecurve was generated by non-linear regression and the EC50 value of eachcurve, which corresponds to the apparent dissociation constant (Kd) ofeach antibody, was calculated using GraphPad Prism v4 (GraphPadsoftware, San Diego, Calif.). A strong shift in fluorescence wasobserved for all antibodies tested and the Kd values correspond to 0.52nM, 1.7 nM, 2.7 nM, 1.1 nM or 0.91 nM for muCD37-3, muCD37-8, muCD37-10,muCD37-12 or muCD37-14 antibodies, respectively.

Likewise, strong binding was also observed when CD37-positive BJABlymphoma cells were used for the same flow cytometry assay describedabove. The Kd values were calculated as described above and correspondto 0.2 nM, 0.4 nM, 0.6 nM, 0.4 nM and 1 nM for muCD37-3, muCD37-38,muCD37-50, muCD37-51, muCD37-56 and muCD37-57, respectively.

Example 3 Pro-Apoptotic Activity of Murine Antibodies

The murine anti-CD37 antibodies induced apoptosis of Ramos and Rajilymphoma cell lines. The degree of apoptosis was measured by flowcytometry analysis after staining with FITC conjugates of Annexin-V(Invitrogen) and with TO-PRO-3 (Invitrogen). In healthy, normal cells,phosphatidylserine is expressed on the inside of the membrane bilayer,and the transition of phosphatidylserine from the inner to the outerleaflet of the plasma membrane is one of the earliest detectable signalsof apoptosis. Annexin V binds phosphatidylserine on the outside but noton the inside of the cell membrane bilayer of intact cells. The degreeof Annexin V binding is therefore an indicator of the induction ofapoptosis. TO-PRO-3 is a monomeric cyanine nucleic acid stain that canonly penetrate the plasma membrane when the membrane integrity isbreached, as occurs in the later stages of apoptosis. Three populationsof cells are distinguishable in two-color flow cytometry: Non-apoptoticcells (Annexin-V negative and TO-PRO-3 negative), early apoptotic cells(Annexin-V positive and TO-PRO-3 negative) and necrotic cells or lateapoptotic cells (Annexin-V positive and TO-PRO-3 positive).

Exponentially growing cells were plated at about 2×10⁵ cells/mL in24-well plates in RMPI-1640 medium supplemented with 10% fetal bovineserum (FBS), 2 mM L glutamine, and 50 μg/mL gentamycin (denoted below ascomplete RMPI-1640 medium). Cells were generally grown in completeRMPI-1640 medium, unless stated otherwise. Cells were incubated with 10nM of anti-CD37 antibodies for 20 to 24 h at 37° C. in a humidified 5%CO₂ incubator. The cells were then pelleted, washed twice with 500 μlPBS, resuspended in 100 μL binding buffer (10 mM Hepes-NaOH, pH 7.4, 140mM NaCl, 2.5 mM CaCl2), and stained with 5 μL of Annexin V-FITC for 15min on ice. Then, 400 μL of binding buffer with 1 μM of TO-PRO-3 wasadded to the mix, and the cell-associated fluorescence of FITC andTO-PRO-3 was immediately measured by flow cytometry. Five thousandevents were collected for each sample. The dot plots for fluorescence ofTO-PRO-3 (FL4-H; y-axis) and fluorescence of Annexin V-FITC (FL1-H;x-axis) were generated using BD CellQuest software.

The percentage of Annexin-V positive cells (includes both TO-PRO-3positive and negative cells) were determined for each sample from theseplots and are shown in FIG. 4 for Ramos cells. Several antibodiesisolated from our antibody screen were tested for pro-apoptotic activityin comparison to rituximab. Unexpectedly, some of the isolated murineanti-CD37 antibodies, such as muCD37-3 and muCD37-12, showed very strongpro-apoptotic activity. Approximately 39% of Ramos cells exposed tomuCD37-3 and 46% of Ramos cells exposed to muCD37-12 were Annexin-Vpositive. In contrast, treatment with the anti-CD20 antibody rituximabresulted in only 13% of Annexin-V positive cells, while untreatedcontrol samples contained 5% Annexin-V positive cells. Several of theisolated murine anti-CD37 antibodies did not show any pro-apoptoticactivity. For example, treatment of Ramos cells with muCD37-8, muCD37-10or muCD37-14 resulted in a minor or no increase in the percentage ofAnnexin-V positive as compared to untreated cells. This is in spite oftheir comparable binding affinity to CD37 as seen in FIG. 3.

Additional antibodies were isolated and screened for their ability toinduce apoptosis in Ramos cells. Of many antibodies isolated that boundCD37 with high affinity, only some had pro-apoptotic activity. Theresults of a Annexin-V assay are shown in FIG. 4B. The murine antibodiesmuCD37-38, muCD37-50, muCD37-51, muCD37-56 and muCD37-57 were able toinduce apoptosis and resulted in 38-45% of Annexin-V positive Ramoscells as compared with 5% in untreated control samples. Similar to theprevious assay, treatment with the anti-CD20 antibody rituximab resultedin only 18% Annexin-V positive cells.

In addition, the murine antibodies were tested their ability to induceapoptosis in Raji lymphoma cells. As seen for Ramos cells, of the manyantibodies isolated that bound CD37 with high affinity, only some hadpro-apoptotic activity. Treatment with muCD37-3 or muCD37-12 resulted in36% or 49% Annexin-V positive cells, respectively. In contrast,treatment with the anti-CD20 antibody rituximab resulted in only 20% ofAnnexin-V positive cells, while untreated control samples contained 4%Annexin-V positive cells.

Likewise, approximately 60% of Raji cells treated with muCD37-3,muCD37-38, muCD37-50, muCD37-51, muCD37-56 or muCD37-57 were Annexin-Vpositive cells compared to 15% of untreated cells.

Example 4 Proliferation Assays

The ability of anti-CD37 antibodies to inhibit cell growth was measuredusing in vitro cytotoxicity assays. Target cells were plated at 5,000cells per well in 100 μL in complete RPMI media (RPMI-1640, 10% fetalbovine serum, 2 mM glutamine, 1% penicillin-streptomycin, all reagentsfrom Invitrogen). Antibodies were diluted into complete RPMI media using3-fold dilution series and 100 μL were added per well. The finalconcentration typically ranged from 3×10⁻⁸ M to 4.6×10⁻¹² M. Cells wereincubated at 37° C. in a humidified 5% CO₂ incubator for 4 to 5 days.Viability of remaining cells was determined by colorimetric WST-8 assay(Dojindo Molecular Technologies, Inc., Rockville, Md., US). WST-8 isreduced by dehydrogenases in living cells to an orange formazan productthat is soluble in tissue culture medium. The amount of formazanproduced is directly proportional to the number of living cells. WST-8was added to 10% of the final volume and plates were incubated at 37° C.in a humidified 5% CO2 incubator for an additional 2-4 hours. Plateswere analyzed by measuring the absorbance at 450 nm (A450) in amultiwell plate reader. Background A450 absorbance of wells with mediaand WST-8 only was subtracted from all values. The percent viability wascalculated by dividing each treated sample value by the average value ofwells with untreated cells. Percent viability=100*(A450 treatedsample−A450 background)/(A450 untreated sample−A450 background). Thepercent viability value was plotted against the antibody concentrationin a semi-log plot for each treatment.

The results from a typical proliferation assay using murine CD37antibodies and SU-DHL-4 lymphoma cells are presented in FIG. 5. It isapparent, that several murine antibodies were able to inhibitproliferation of SU-DHL-4 cells substantially and in a dose-dependentmanner, while others had no such effect. For example, treatment withmuCD37-3 reduced the cell viability to 34% at the highest antibodyconcentration tested with an EC50 of 0.17 nM. Similarly, treatment withmuCD37-38 reduced the cell viability to 25% at the highest antibodyconcentration tested with an EC50 of 0.19 nM. Likewise, treatment withmuCD37-50 or muCD37-51 reduced the cell viability to 38% at the highestantibody concentration tested with an EC50 of 0.25 nM or 0.5 nM,respectively. In contrast, treatment with for example CD37-16 did notreduce cell viability in a dose-dependent manner.

Example 5 Cloning and Sequencing of the VL and VH Regions of the CD37-3Antibody

Total cellular RNA was prepared from 5×10⁶ cells of the CD37-3 hybridomausing an RNeasy kit (QIAgen) according to the manufacturer's protocol.cDNA was subsequently synthesized from total RNA using the SuperScriptII cDNA synthesis kit (Invitrogen).

The procedure for the first round degenerate PCR reaction on the cDNAderived from hybridoma cells was based on methods described in Wang etal. ((2000) J Immunol Methods. 233:167-77) and Co et al. ((1992) JImmunol. 148:1149-54). VH sequences were amplified by PCR using thefollowing degenerate primers: EcoMH1 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC(SEQ ID NO:171), EcoMH2 CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ IDNO:172) and BamIgG1 GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC (SEQ IDNO:173). VL sequences were amplified by PCR using the followingdegenerate primers: SacIMK GGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ IDNO:174) and HindKL TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC (SEQID NO:175). (Mixed bases are defined as follows: N=G+A+T+C, S=G+C,Y=C+T, M=A+C, R=A+G, W=A+T). The PCR reaction mixtures were then run ona 1% low melt agarose gel, the 300 to 400 bp bands were excised,purified using Zymo DNA mini columns, and sent to Agencourt Biosciencesfor sequencing. The respective 5′ and 3′ PCR primers were used assequencing primers to generate the variable region cDNAs from bothdirections. The amino acid sequences of VH and VL regions were predictedfrom the DNA sequencing results.

Since the degenerate primers used to clone the VL and VH cDNA sequencesalters the 5′ end sequences, additional sequencing efforts were neededto verify the complete sequences. The preliminary cDNA sequences wereused to search the NCBI IgBlast site(http://www.ncbi.nlm.nih.gov/igblast/) for the murine germline sequencesfrom which the antibody sequences are derived. PCR primers were thendesigned to anneal to the germline linked leader sequence of the murineantibody so that this new PCR reaction would yield a complete variableregion cDNA sequence, unaltered by the PCR primers. The PCR reactions,band purifications, and sequencing were performed as described above.

Mass Determination for Sequence Confirmation

The cDNA sequence information for the variable region was combined withthe germline constant region sequence to obtain full length antibodycDNA sequences. The molecular weights of the heavy chain and light chainwere then calculated and compared with the molecular weights obtained byLC/MS analyses of the murine CD37-3 antibody. The molecular weightmeasurements are consistent with the cDNA sequences for both the CD37-3light and heavy chain.

Chimerization

The variable sequence for the light chain variable region is cloned intoEcoRI and BsiWI sites in the pchCD37-3LCZ plasmid. The heavy chainvariable region is cloned into the HindIII and Apa1 sites in thepchCD37-3HCN plasmid. Equivalent plasmids were constructed forchCD37-12. These plasmids were used to express chimeric antibodies inHEK-293T cells using a standard calcium phosphate procedure (BDBiosciences, CalPhos Mammalian Transfection Kit, Cat #631312).Supernatant was purified using standard Protein A chromatographyprocedures as described above, but the polishing chromatography stepswere performed using either carboxymethyl (CM) fast flow ion exchange(IEX) resin (GE Lifesciences) and 10 mM potassium phosphate, 10 mMsodium chloride binding buffer (pH 7.5) or the alternative CHT methodsdescribed above.

Example 6 Antibody Humanization

The CD37-3 and huCD37-50 antibodies were humanized following resurfacingmethods previously described, such as, for example in Roguska et al.,Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994) and Roguska et al.,Protein Eng. 9(10):895-904 (1996), which are incorporated in theirentirety herein by reference. Resurfacing generally involvesidentification of the variable region framework surface residues in bothlight and heavy chains and replacing them with human equivalents. Themurine CDR's are preserved in the resurfaced antibody. Exemplary CDRs ofCD37-3 and CD37-50 are defined as indicated in Table 11. In addition tothe heavy chain CDR2 definition employed for resurfacing, the tableprovides exemplary Kabat defined heavy chain CDR2's for both the murineand human CD37-3 and CD37-50. The underlined sequence marks the portionof the Kabat heavy chain CDR2 not considered a CDR for resurfacing.

TABLE 11 CD37-3 CDR's CD37-50 CDR's Light Chain Light Chain CDR1:  CDR1:RASENIRSNLA SATSSVTYMH (SEQ ID NO: 28) (SEQ ID NO: 37) CDR2:Murine CDR2: VATNLAD DTSKLPY (SEQ ID NO: 29) (SEQ ID NO: 38) CDR3:Human CDR2: YWGTTWT DTSNLPY (SEQ ID NO: 30) (SEQ ID NO: 40) CDR3:QQWSDNPPT (SEQ ID NO: 39) Heavy Chain Heavy Chain CDR1: CDR1: TSGVSSGFAWH (SEQ ID NO: 4) (SEQ ID NO: 13) CDR2:  CDR2: VIWGDGSTN YILYSGSTV(SEQ ID NO: 5) (SEQ ID NO: 14) CDR3: CDR3: GGYSLAH GYYGYGAWFAY(SEQ ID NO: 6) (SEQ ID NO: 15) Kabat Defined CD37-3Kabat Defined CD37-50  HC CDR2 HC CDR2 Murine HC CDR2: Murine HC CDR2:VIWGDGSTNYHSALKS YILYSGSTVYSPSLKS (SEQ ID NO: 176) (SEQ ID NO: 178)Human HC CDR2: Human HC CDR2: VIWGDGSTNYHPSLKS YILYSGSTVYSPSLKS(SEQ ID NO: 177) (SEQ ID NO: 179)

The CD37-3 and CD7-50 light and heavy chain CDR's as defined for theresurfacing are given by way of example in Table 11. Lysine 53 in murineCD37-50 light chain CDR2 was replaced with asparagine in humanizedCD37-50 (shown in italic) so both versions of the LC CDR2 are given. TheKabat definition for heavy chain CDR2 is also given for both the murineand human CD37-3. The underlined sequence marks the portion of the Kabatheavy chain CDR2 not considered a CDR for resurfacing.

Surface residue positions are defined as any position with its relativeaccessibility of 30% or greater (Pedersen J. T. et. Al, J. Mol. Biol.1994; 235: 959-973). Surface residues are then aligned with humangermline surface sequences to identify the most homologous human surfacesequence. For CD37-3, the human germline sequences used as thereplacement surfaces were IGKV1/OR2-0*01 and IGHV4-34*09 for VL and VH,respectively. For CD37-50, the human germline sequences used as thereplacement surfaces were IGKV3/OR2-268*01 and IGHV4-31*03 for VL andVH, respectively. As can be seen from the lists in FIG. 6, a total ofseven surface residues in the light chain and seven in the heavy chainwere replaced with the human counterparts in CD37-3. As seen in FIG. 7for CD37-50, the total surface residues that were replaced with humancounterparts are seven and five in VL and VH, respectively. In CD37-3,the heavy chain residue 61 is in close proximity to CDR-H2 and since itssubstitution to the human residue proline might result in reducedbinding affinity, a second resurfaced version was generated with murineserine residue retained. Since these antibodies were being tested ascytotoxic conjugates, the CD37-50 light chain CDR2 lysine 53 wasreplaced with an asparagine to avoid the concerns that lysineconjugation could impact binding affinity. FIG. 8 shows the alignment ofthe resurfaced sequences for the CD37-3 and CD37-50 variable domain ofboth light chain and heavy chain with their murine counterparts.

Recombinant Expression of huCD37-3 Antibody

The variable region sequences for huCD37-3 and CD37-50 werecodon-optimized and synthesized by Blue Heron Biotechnology. Thesequences are flanked by restriction enzyme sites for cloning in-framewith the respective constant sequences in single chain mammalianexpression plasmids. The light chain variable region is cloned intoEcoRI and BsiWI sites in the pAbKZeo plasmid. The heavy chain variableregion is cloned into the HindIII and Apa1 sites in the pAbG1Neoplasmid. These plasmids can be used to express the recombinantantibodies in either transient or stable mammalian cell transfections.Transient transfections to express recombinant antibodies in HEK 293Tcells were performed using a modified PEI procedure (Durocher, Y. etal., Nucleic Acids Res. 30:E9 (2002)). Supernatant was purified byProtein A and polishing chromatography steps using standard proceduresas described above for chimerized antibodies.

Expression of TRU-016

In order to compare the activity of the isolated anti-CD37 antibodies,previously identified anti-CD37 antibodies were cloned and expressed.The DNA sequence for the anti-CD37 SMIP was drawn from US2007/0059306using SEQ ID 51. The sequence was flanked by HindIII and Xho1restriction enzyme sites for cloning into the pAbG1Neo mammalianexpression plasmids. Expression and purification was carried out asdescribed for huCD37-3 above.

Example 7 Binding Affinity of Chimeric Antibodies

The chimeric antibodies chCD37-3 and chCD37-12 were assayed for theirbinding affinity to Ramos cells in comparison to their murinecounterparts. Flow cytometry binding assays using Ramos cells andmuCD37-3, chCD37-3, muCD37-12 and chCD37-12 antibodies were carried outand analyzed as described in Example 2 using secondary FITC-conjugatedgoat-anti-murine and -anti-human antibodies. FIG. 9A depicts thedose-response curves generated by non-linear regression for eachantibody. The value for the apparent dissociation constant (Kd) of eachantibody was calculated using GraphPad Prism v4 (GraphPad software, SanDiego, Calif.). It is apparent that chimerization did not greatly affectthe binding affinity of either antibody as the Kd for muCD37-3,chCD37-3, muCD37-12 and chCD37-12 corresponds to 0.4 nM, 0.8 nM, 0.8 nMand 1.2 nM, respectively.

Binding Affinity of huCD37-3v1.0 and huCD37-3v1.01

Flow cytometry binding assays using BJAB cells and a competitive bindingformat were used to evaluate binding affinity of chimeric and humanizedversions of CD37-3. BJAB cells were incubated with a 1 nM concentrationof PE-labeled muCD37-3 antibody and competition was measured by addingvarying amounts of muCD37-3, chCD37-3, huCD37-3v1.0 or huCD37-3v1.01.The samples were incubated for 3 hrs at 4° C. Then, the cells werepelleted, washed with FACS buffer and resuspended in 200 μL of PBScontaining 1% formaldehyde. Samples were acquired using a FACSCaliburflow cytometer with the HTS multiwell sampler and analyzed usingCellQuest Pro (all from BD Biosciences, San Diego, US). The resultingmean PE fluorescence was plotted against the amount of competingantibody used in a semi-log plot. FIG. 9B depicts the dose-responsecurves generated by non-linear regression for each antibody. The valuefor the apparent dissociation constant (Kd) of each antibody wascalculated using GraphPad Prism v4 (GraphPad software, San Diego,Calif.). It is apparent that chimerization or humanization did notaffect the binding affinity of CD37-3 as all version compete equallywell for binding with the murine parent antibody. The EC50 ofcompetition binding for muCD37-3, chCD37-3, huCD37-3v1.0 orhuCD37-3v1.01 corresponds to 0.8 nM, 0.7 nM, 1 nM and 0.6 nM,respectively.

Binding Affinity of Humanized Antibodies

The humanized antibodies huCD37-38, huCD37-50, huCD37-51, huCD37-56 andchCD37-57 were assayed for their binding affinity to BJAB cells incomparison to their murine counterparts. Flow cytometry binding assayswere carried out using secondary FITC-conjugated goat-anti-murine and-anti-human antibodies, analyzed as described in Example 2 anddose-response curves were generated by non-linear regression for eachantibody. The value for the apparent dissociation constant (Kd) of eachantibody was calculated using GraphPad Prism v4 (GraphPad software, SanDiego, Calif.). It is apparent that humanization did not greatly affectthe binding affinity of any antibody. The Kd for muCD37-3 and huCD37-3corresponds to 0.2 nM, while the Kd for muCD37-38 and huCD37-38corresponds to 0.4 nM and 0.3 nM, respectively. Similarly, the Kd formuCD37-50 and huCD37-50 corresponds to 0.6 nM and 0.2 nM, respectively,while the Kd for muCD37-51 and huCD37-51 corresponds to 0.6 nM and 0.8nM, respectively. Finally, the Kd for muCD37-56 and huCD37-56corresponds to 0.4 nM and 0.2 nM, respectively, while the Kd formuCD37-57 and huCD37-57 corresponds to 1.0 nM and 0.3 nM, respectively.

Example 8 Expression of Macaque CD37

The CD37 AA sequence of macaque CD37 was obtained from Genbank (G1:718718). The sequence was codon-optimized and synthesized by Blue HeronBiotechnology. An expression plasmid pSRa-CD37mac was constructed thatcontained the entire CD37 coding sequence (CDS) from macaque flanked byXbaI and BamHI restriction sites that allowed expression of macaqueCD37. 300-19 cells, a pre-B cell line derived from a Balb/c mouse (M. G.Reth et al. 1985, Nature, 317: 353-355), was transfected with thisexpression plasmid to stably express macaque CD37 on the cell surface.

Binding Affinity of Murine Antibodies to Macaque CD37

The murine antibodies muCD37-3, muCD37-12, muCD37-38, huCD37-50,muCD37-51, muCD37-56 and muCD37-57 were assayed for their ability tobind to 300-19/CD37mac cells expressing macaque CD37. Flow cytometrybinding assays were carried out using secondary FITC-conjugatedgoat-anti-murine or goat-anti-human antibodies, analyzed as described inExample 2. Binding was compared to the previously described anti-CD37antibody WR17 and the anti-CD37 SMIP TRU-016. As can be seen from FIG.10A, several isolated anti-CD37 antibodies, muCD37-38, huCD37-50,muCD37-51, muCD37-56 and muCD37-57 can bind to the macaque derived CD37antigen. In contrast, muCD37-3, muCD37-12, the previously describedanti-CD37 antibody WR17 and the anti-CD37 SMIP TRU-016 are unable tobind the macaque derived CD37 antigen.

Binding Affinity of Humanized Antibodies to Macaque CD37

The humanized antibodies huCD37-38, huCD37-50, huCD37-51, huCD37-56 andhuCD37-57 were assayed for their binding affinity to 300-19/CD37maccells expressing macaque CD37. Flow cytometry binding assays werecarried out using secondary FITC-conjugated goat-anti-human antibodies,analyzed as described in Example 2 and dose-response curves weregenerated by non-linear regression for each antibody. The value for theapparent dissociation constant (Kd) of each antibody was calculatedusing GraphPad Prism v4 (GraphPad software, San Diego, Calif.). It isapparent from FIG. 10B, that several isolated humanized antibodies bindto macaque CD37 while huCD37-3 does not. The Kd value for huCD37-38,huCD37-50, huCD37-51, huCD37-56 and huCD37-57 correspond to 1.1 nM, 1.8nM, 14 nM, 5 nM, and 2 nM, respectively. Therefore, humanization doesnot affect the binding specificity of the isolated antibodies.

Example 9 Pro-Apoptotic Activity of Chimeric and Humanized Antibodies

The pro-apoptotic activity of chimeric and humanized antibodies wasevaluated on Ramos cells. Cells were incubated with 10 nM concentrationof antibodies or an huIgG isotype control antibody for 20 hrs followedby Annexin-V-FITC and TO-PRO-3 staining and flow cytometry analysis.ChCD37-12 retained strong pro-apoptotic activity of muCD37-12.Approximately 40% of Ramos cells are Annexin-V positive after treatmentwith either muCD37-12 or chCD37-12 antibody as compared to 4% ofuntreated control cells. Similarly, approximately 40% of Ramos cells areAnnexin-V positive after treatment with huCD37-3, huCD37-38, huCD37-50,huCD37-51, huCD37-56 or huCD37-57 antibody as compared to 4% of isotypecontrol treated or untreated cells. In contrast, treatment with theanti-CD20 antibody rituximab resulted in only 13% of Annexin-V positivecells. This result demonstrates that the strong pro-apoptotic activityof the murine anti-CD37 antibodies isolated here is retained by thechimeric or humanized antibodies derived from them. Therefore, theunique functional property of this group of anti-CD37 antibodies, strongpro-apoptotic activity in the absence of cross-linking, is notnegatively affected by chimerization or humanization.

Pro-Apoptotic Activity of huCD37-3 and TRU-016

The pro-apoptotic activity of huCD37-3 against Ramos and Raji lymphomacells was compared to the anti-CD37 SMIP TRU-016. TRU-016 has beendescribed as a compound with no pro-apoptotic activity unlesscross-linked with a secondary antibody. It is apparent that exemplaryanti-CD37 antibodies huCD37-3 and chCD37-38 have much strongerpro-apoptotic activity against both lymphoma cell lines. Treatment withhuCD37-3 or chCD37-38 resulted in 40% or 49% Annexin-V positive Ramoscells, as compared to 3% of untreated control cells. Rituximab treatmentresulted in only 15% Annexin-V positive Ramos cells. In contrast,TRU-016 treatment did not increase the percentage of Annexin-V positiveRamos cells. Likewise, Treatment with huCD37-3 or chCD37-38 resulted in34% or 30% Annexin-V positive Raji cells, as compared to 7% of untreatedcontrol cells. Rituximab treatment resulted in only 19% Annexin-Vpositive Raji cells. In contrast, TRU-016 treatment did not increase thepercentage of Annexin-V positive Ramos cells.

Dose Response for Pro-Apoptotic Activity of Humanized Antibodies

Varying amounts of each antibody were incubated with Ramos cells for 20hrs followed by Annexin-V-FITC and TO-PRO-3 staining and flow cytometryanalysis. The percentage of Annexin-V positive cells was plotted againstthe antibody concentration in a semi-log plot, and EC50 values werecalculated from curves fitted using non-linear regression analysis. Itis apparent that all humanized antibodies have strong pro-apoptoticactivity with a maximum percentage of Annexin-V positive cells of atleast 40%. FIG. 11. The EC50 for this activity corresponds to 0.08, 0.08and 0.11 nM for huCD37-3, huCD37-38 and huCD37-50, respectively. Inaddition, the EC50 for this activity corresponds to 0.41, 0.57 and 1.01nM for huCD37-51, huCD37-56 and huCD37-57, respectively. In contrast,treatment with the anti-CD20 antibody rituximab resulted in a maximumpercentage of Annexin-V positive cells of only 15% compared with 4% ofcells treated with isotype control antibody.

Example 10 Proliferation Assays for Chimeric and Humanized Anti-CD37Antibodies

The ability of chimeric and humanized anti-CD37 antibodies to inhibitcell growth was measured using in vitro cytotoxicity assays as describedin Example 4. The results from a typical proliferation assay usingSU-DHL-4 and DOHH-2 lymphoma cells are presented in FIG. 12. It isapparent, that all antibodies were able to inhibit proliferation ofSU-DHL-4 cells substantially and in a dose-dependent manner. Forexample, treatment with muCD37-3 reduced the viability of SU-DHL-4 cellsto 35% with an EC50 of 0.07 nM. Similarly, treatment with chCD37-3,huCD37-3v1.0 or huCD37-3v1.01 reduced the viability of SU-DHL-4 cells toapproximately 30% at the highest antibody concentration tested with anEC50 of 0.03 nM, 0.06 nM or 0.03 nM, respectively. Likewise, allantibodies were able to inhibit proliferation of DOHH-2 follicularlymphoma cells substantially and in a dose-dependent manner. Forexample, treatment with muCD37-3 reduced the viability of DOHH-2 cellsto 45% with an EC50 of 0.05 nM. Similarly, treatment with chCD37-3,huCD37-3v1.0 or huCD37-3v1.01 reduced the viability of DOHH-2 cells toapproximately 35% with an EC50 of 0.06 nM, 0.07 nM or 0.05 nM,respectively. This result demonstrates that the various version of theCD37-3 antibody have similar anti-proliferative activity that is notaffected by chimerization or humanization.

Additional humanized anti-CD37 antibodies were tested in similar invitro cytotoxicity assays. All humanized antibodies tested were able toinhibit proliferation of SU-DHL-4 cells substantially and in adose-dependent manner. For example, treatment with huCD37-38 reduced theviability of SU-DHL-4 cells to 24% with an EC50 of 0.42 nM, whiletreatment with huCD37-50 reduced the viability of SU-DHL-4 cells to 31%with an EC50 of 0.39 nM. In contrast, treatment with the anti-CD20antibody rituximab reduced the viability of SU-DHL-4 cells to 35% withan EC50 of 1.6 nM. In addition, treatment with huCD37-51 or huCD37-56reduced the viability of SU-DHL-4 cells to 24% with an EC50 of 0.60 nMor 0.68 nM, respectively. Furthermore, treatment with huCD37-57 reducedthe viability of SU-DHL-4 cells to 31% with an EC50 of 0.42 nM.Treatment with an isotype control antibody did not have an effect on theviability of SU-DHL-4 cells.

Anti-Proliferative Activity of huCD37-3 in Comparison to OtherAntibodies

To further characterize the anti-proliferative activity of the isolatedanti-CD37 antibodies, we compared the effect of the exemplary huCD37-3antibody to that of the anti-CD37 SMIP TRU-16 compound.Immunohistochemistry using tumor microarrays confirmed that CD37 andCD20 exhibited similar expression patterns and prevalances in subtypesof NHL. See Table 12 below. Thus, comparisons were also made to theanti-CD20 antibody rituximab. The panel of cell lines includedGranta-519, SU-DHL-4, Namalwa and Daudi lymphoma cells. FIG. 13.

TABLE 12 CD37 staining on lymphoma tumor microarrays in comparison toCD20 staining. # of pos. cores (≧1 hetero) # total Tumor histology CD37CD20 cores T-cell lymphoma 0 0 4 Multiple myeloma 0 0 10 Hodgkin'sB-cell lymphoma 1 (8%) 1 (8%) 12 Non-Hodgkin B cell 21 (95%) 21 (95%) 22lymphoma (unspecified) Follicular lymphoma 3 (100%) 3 (100%) 3 MALTlymphoma 3 (100%) 3 (100%) 3 Diffuse large B cell lymphoma 13 (93%) 13(93%) 14 Burkitt's lymphoma 6 (75%) 7 (88%) 8 Mantle cell lymphoma 3(50%) 6 (100%) 6

In all case, huCD37-3 treatment resulted in a reduction in cellviability in a dose-dependent manner. For example, treatment withhuCD37-3 reduced the viability of Granta-519 cells to approximately 37%with an EC50 of 0.062 nM. Rituximab treatment reduced the viability ofGranta-519 cells to approximately 47% with an EC50 of 0.36 nM. Treatmentwith huCD37-3 reduced the viability of SU-DHL-4 cells to approximately17% with an EC50 of 0.053 nM. Rituximab treatment reduced the viabilityof SU-DHL-4 cells to approximately 20% with an EC50 of 0.2 nM. Instriking contrast, treatment with TRU-016 did not reduce the viabilityof Granta-519 or SU-DHL-4 cells to a significant degree or in adose-dependent manner. In further examples, treatment with huCD37-3reduced the viability of Namalwa cells to approximately 47% with an EC50of 0.1 nM and reduced the viability of Daudi cells to approximately 68%with an EC50 of 0.25 nM. Rituximab treatment did not have an effect onNamalwa cells but reduced the viability of Daudi cells to approximately69% with an EC50 of 2.6 nM. In striking contrast, treatment with TRU-016did not reduce the viability of Namalwa or Daudi cells to a significantdegree or in a dose-dependent manner. Finally, treatment with huCD37-3reduced the viability of Ramos cells to approximately 53% with an EC50of 0.08 nM, while neither TRU-016 nor rituximab treatment had any effecton Ramos cell viability. This result underscores the uniqueness of theanti-proliferative activity of the isolated anti-CD37 antibodies.

Example 11 CDC Activity of CD37 Antibodies

To assess complement-dependent cytotoxicity (CDC) activities of chimericand humanized anti-CD37 antibodies, cell based assays were performedaccording to a published method (Gazzano-Santoro H, J Immunol Methods.1997 202(2):163-71). Antibodies were aliquoted in duplicate at 50μL/well into a flat-bottom 96-well tissue culture plate at variousconcentrations typically ranging from 5 μg/mL (=3.3×10⁻⁸ M) to 2.3 ng/mL(=1.5×10⁻¹¹ M) in RHBP (RPMI-1640, 20 mM HEPES, 0.1% BSA, 1%penicillin-streptomycin) medium. Target cells were added to theantibodies at 5×10⁴ cells in 100 μL of RHBP medium per well. Lyophilizedhuman complement (Sigma-Aldrich, St. Louis, US) was reconstituted with 1mL sterile purified water per vial and diluted 5-fold to a 20% stockwith RHBP media immediately before use. 50 μL/well of complementsolution was added to each well for a final concentration of 5%. Plateswere incubated for 2 h at 37° C. in 5% CO₂ humidified incubator to allowfor complement mediated lysis. After this incubation time, Alamar Bluereagent (Invitrogen) was added to each well at a final concentration of10% to measure the viability of the remaining cells. The plate wasincubated for 16 to 20 hours at 37° C. before measuring the fluorescence(in relative fluorescence units, RFU) at EX540/EM590 nm. Controlsincluded triplicate wells with media and complement but without cells(media only, 0% viability) and wells with cells and complement butwithout antibody (cells only, 100% viability). The percentage ofspecific cell viability for each sample was determined by to thefollowing formula: Percent viability=(sample−media only)/(cellsonly−media only).

The result of an exemplary CDC assay using Ramos cells is presented inFIG. 14. Strikingly, chCD37-12 has potent CDC activity against Ramoscells. It reduced the viability of Ramos cells to 32% at the highestantibody concentration tested with an EC50 of 0.037 μg/mL. In addition,several isolated antibodies showed CDC activity against Ramos to avarying degree. Treatment with huCD37-3, huCD37-38, huCD37-50 resultedin a reduction in cell viability to 59%, 50% and 45%, respectively.Treatment with huCD37-51, huCD37-56 or huCD37-56 moderately reduced cellviability of Ramos cells to approximately 70-80% at the highest antibodyconcentration tested.

Example 12 ADCC Activity of CD37 Antibodies

A lactate dehydrogenase (LDH) release assay was used to measureantibody-dependent cell mediated cytotoxicity (ADCC) of tumor cellslines using freshly isolated human natural killer (NK) cells as effectorcells (Shields R L, J Biol Chem. 2001 276(9):6591-604). The NK cellswere first isolated from human blood from a normal donor (Research BloodComponents, Inc., Brighton, Mass.) using a modified protocol for the NKIsolation Kit II (Miltenyi Biotech, 130-091-152). Blood was diluted2-fold with 1×PBS. 25 mL of diluted blood was carefully layered over 25mL of Ficoll Paque in a 50 mL conical tube and centrifuged at 400 g for45 min at RT. The peripheral blood mononuclear cells (PBMC) werecollected from the interface, transferred into a new conical 50 mL tube,and washed once with 1×PBS. The PBMC were resuspended in 2 mL ofNK-isolation buffer (1×PBS, 0.5% BSA, 2 mM EDTA), and then 500 μL ofBiotin-Antibody Cocktail were added to the cell suspension. TheBiotin-Antibody Cocktail contains biotinylated antibodies that bind tothe lymphocytes, except for NK cells, resulting in a negative selectionof NK cells. The mixture was incubated at 4° C. for 10 min, and then 1.5mL of NK-isolation buffer and 1 mL of Anti-Biotin Micro Beads wereadded. The cell-antibody mixture was incubated for another 15 min at 4°C. Next, cells were washed once with 50 mL of NK-isolation buffer andresuspended in 3 mL of NK-isolation buffer. Then, a MACS LS column wasmounted on the autoMACS separator (Miltenyi Biotech) and pre-washed with3 mL of NK-isolation Buffer. The cell suspension was automaticallyapplied onto the column, washed and the effluent fraction with unlabeledNK cells was collected into a new 50-mL conical tube. The resulting NKcells were plated into 30 mL of complete RPMI media (RPMI-1640supplemented with 5% fetal bovine serum, 1% penicillin-streptomycin, 1mM HEPES, 1 mM Sodium Pyruvate, 1% 100×MEM non-essential Amino AcidSolution) overnight. The subsequent assay and all dilutions were carriedout in RHBP medium (RPMI-1640 medium supplemented with 20 mM HEPES, pH7.4, 0.1% BSA and 1% penicillin-streptomycin).

Various concentrations of antibodies in RHBP medium were aliquoted induplicate at 50 μL/well into a round bottom 96-well plate. The targetcells were resuspended at 106 cells/mL in RHBP medium and added at 100μL/well to each well containing antibody dilutions. The plate containingtarget cells and antibody dilutions was incubated for 30 min at 37° C.NK cells were then added to the wells containing the target cells at 50μL/well. The typical ratio was 1 target cell to 3-4 NK cells. Thefollowing controls were set up for each experiment: NK cells alone,target cells alone (spontaneous LDH release), target cells with NK cells(antibody independent LDH release), target cells with 10% Triton X-100(maximum LDH release). The mixtures were incubated at 37° C. for 4 h toallow for cell lysis. Plates were centrifuged for 10 min at 1200 rpm,and 100 μL of the supernatant was carefully transferred to a newflat-bottom 96-well plate. LDH reaction mixture (100 μL/well) from theCytotoxicity Detection Kit (Roche 1 644 793) was added to each well andincubated at room temperature for 5 to 30 min. The optical density ofsamples was measured at 490 nm (OD490). The percent specific lysis ofeach sample was determined using the following formula: percent specificlysis=(sample value−spontaneous release)/(maximum release−spontaneousrelease)*100.

Incubation with humanized antibodies lead to good ADCC activity againstDaudi, Ramos and Granta-519 lymphoma cells in the presence of human NKeffector cells. Their ADCC activity against Daudi lymphoma cells wascompared with the ADCC activity of TRU-016 (FIG. 15).

Treatment with huCD37-3, huCD37-38 or huCD37-50 antibodies resulted inapproximately 41%, 39% or 40% Daudi cell lysis with an EC50 value of0.42 ng/mL, 1.31 ng/mL, or 2.42 ng/mL, respectively. This activity wassimilar to that resulting from TRU-016 treatment with 42% Daudi celllysis observed and an EC50 value of 0.93 ng/mL. In addition, treatmentwith huCD37-51, huCD37-56 and huCD37-57 resulted in approximately 39%,36% or 36% of Daudi cell lysis with an EC50 value of 5.7 ng/mL, 4.3ng/mL, or 7.9 ng/mL, respectively.

The ADCC activity of the isolated antibodies against Ramos lymphomacells was compared with the ADCC activity of TRU-016. Treatment withhuCD37-3, huCD37-38 or huCD37-50 antibodies resulted in approximately43%, 42% or 46% Ramos cell lysis with an EC50 value of 0.95 ng/mL, 2.0ng/mL, or 3.0 ng/mL respectively. This activity was similar to thatresulting from TRU-016 treatment with 59% Ramos cell lysis observed andan EC50 value of 1.53 ng/mL. In addition, treatment with huCD37-51,huCD37-56 and huCD37-57 resulted in approximately 53%, 43% or 44% ofRamos cell lysis with an EC50 value of 5.7 ng/mL, 4.3 ng/mL, or 7.9ng/mL, respectively.

In additional experiments the ADCC activity of huCD37-3 and chCD37-38against Granta-519 cells was compared with the ADCC activity of TRU-016.Treatment with huCD37-3 or chCD37-38 antibodies resulted inapproximately 19% or 18% Granta-519 cell lysis with an EC50 value of0.13 ng/mL, or 0.73 ng/mL respectively. TRU-016 treatment resulted in16% Granta-519 cell lysis observed and an EC50 value of 0.83 ng/mL.

Example 13 Epitope Mapping

The localization of amino acid requirements for the epitopes ofdifferent CD37 antibodies can help tie common or unique functionalcharacteristics to specific molecular interactions. The extracellulardomain of CD37 contains two extracellular loops, a small loop of about18 residues, and a larger one consisting of approximately 135 aminoacids. Epitope requirements have not been described for previouslypublished CD37 antibodies. To further characterize the isolated CD37antibodies of this invention, we constructed several CD37 antigenvariants with AA substitution in the larger extracellular loop.

CD37 Variant Cloning and Expression

Mammalian expression plasmids were built containing either the entirehuman or macaca CD37 cDNA sequences, codon optimized and synthesized byBlue Heron Biotechnologies, and flanked by XbaI and BamHI restrictionsites to facilitate cloning into the pSRa vector multiple cloning site.Since the human and macaca CD37 sequences are highly homologous (FIG.16), the expression of these constructs could distinguish the human andmacaca cross reactive antibodies from those that recognize epitopesrequiring at least one of the 11 extracellular CD37 amino aciddifferences between in these two species as described in Example 8.

To further characterize the CD37 antibody epitopes, a series of murineand human chimeric CD37 constructs were built. Because of the size andhigh homology of the small CD37 extracellular loop, epitope localizationefforts were limited to the large extracellular loop. An EcoRV to Pst1cassette encoding residues 1108 to Q235 of the large extracellular loopwas redesigned to be further segmented into 5 sections by incorporating4 unique restriction sites that could be conserved between the human,murine, and macaca CD37 sequences (FIG. 17). The following human andmouse chimeric CD37 constructs were created:

hCD37-M1 (SEQ ID NO: 180)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37-M2 (SEQ ID NO: 181)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37-M3 (SEQ ID NO: 182)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNSTATNDSTVFDKLFFSQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37-M45 (SEQ ID NO: 183)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR muCD37-R176 (SEQ ID NO: 184)ISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQ.

In order to preserve one such restriction site, Kpn1, the human R176 wasincluded in all of the murine/human chimeric constructs. The murine andhuman CD37 cassettes were ordered from Blue Heron and cloned into thepSRa vector together with a 5′ end fragment of the original human CD37construct generated by PCR to incorporate the EcoRV site and the 3′ endPst1 to Xba1 restriction fragment taken from the original macaca CD37construct. The murine and human chimeric CD37 cassettes were then builtusing standard restriction digests and ligations taking advantage of thecommon unique restriction sites.

The hCD37-M1 variant was created by inserting a EcoRV-SacII restrictionfragment encoding the AA 5109 to A138 of the analogous murine CD37sequence. Likewise the hCD37-M3 variant was created by inserting aKpnI-B1 μl restriction fragment encoding the AA V177 to L201 of theanalogous murine CD37 sequence. The hCD37-M45 variant was created byinserting a BlpI-PstI restriction fragment encoding the AA 5202 to 1243of the analogous murine CD37 sequence. The resulting clones wereverified by restriction enzyme digestion followed by DNA sequencing.

Stable cell lines were obtained by transfection of the murine and humanchimeric CD37 variant expression plasmids into 300-19 cells usingstandard electroporation procedures. Briefly, 5×10⁶ 300-19 cells wereelectroporated in cold RPMI-1640 media using a BioRad Gene Pulser set at260V and 960 μF. Subsequently, cells were diluted and plated into96-well plates in RPMI-1640 media supplemented with 10% FBS and 50 μMβ-mercaptoethanol. After 24 hours G418 (Invitrogen) was added at a finalconcentration of 2 mg/mL to select for transfected cells. After 2 weeks,single colonies were isolated, analyzed for CD37 surface expression byflow cytometry and expanded.

Antibody Binding to CD37 Variants

Binding of various CD37 antibodies to cells expressing human CD37wildtype and variants was analyzed by flow cytometry using 1.5 μg/mL ofeach antibody. The isolated antibodies of this invention were comparedto commercially available CD37 antibody WR17, as well as the TRU-016SMIP. As can be seen in FIG. 18A, all antibodies bound to wild type CD37expressing cells. Likewise, all antibodies tested bound the hCD37-M3variant (FIG. 18B). In contrast, the isolated antibodies of thisinvention bound the hCD37-M1 variant, while TRU-016 and WR17 were unableto bind hCD37-M1 variant (FIG. 19A). The CD37-50 and CD37-51 antibodiesand TRU-016 were also able to bind the hCD37-M45 variant. WR17 showedpartial binding to the hCD37-M45 variant, while the other antibodiesCD37-3, CD37-12, CD37-38, CD37-56 and CD37-57 were unable to bind (FIG.19B). This suggests that all of the isolated antibodies of thisinvention do not require the 12 AA residues in the hCD37-M1 variant thatwere changed to the corresponding murine AA residues for binding to theCD37 antigen. In contrast, the CD37-3, CD37-12, CD37-38, CD37-56 andCD37-57 antibodies require at least one of the AA residues in thehCD37-M45 variant that were changed to the corresponding murine AAresidues for binding to the CD37 antigen.

This unexpected result indicates that the isolated antibodies of thisinvention represent a novel class of CD37 antibodies with a uniquecombination of functional characteristics.

In addition, similar constructs are built following the same design. Theconstructs contain various combinations of murine, human and/or macacasequences encoding the large extracellular loop of CD37 (see FIG. 17).Examples of constructs with a single human section inserted into themurine large extracellular loop sequence are:

hCD37mECD-H1: (SEQ ID NO: 185)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARY hCD37mECD-H2: (SEQ ID NO: 186)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37mECD-H3: (SEQ ID NO: 187)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNLSATNDSTILDKVILPQLSRLGPRAKLRQTADICALPAKAHIYREGCAQSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37mECD-H4: (SEQ ID NO: 188)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGHLARSRHSADICALPAKAHIYREGCAQSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37mECD-H5 (SEQ ID NO: 189)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGPRAKLRQTADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR and hCD37mECD-H45(SEQ ID NO: 190) MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRVRLERRVQELVLRTIQSYRTNPDETAAEESWDYAQFQLRCCGWQSPRDWNKAQMLKANESEEPRVPCSCYNSTATNDSTVFDKLFFSQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR.

Further examples are constructs with a single macaca section insertedinto the human large extracellular loop sequence such as:

hCD37-Mac12: (SEQ ID NO: 191)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLQDIVEKTIQRYHTNPEETAAEESWDYVQFQLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37-Mac4: (SEQ ID NO: 192)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPAESHIYREGCAQGLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR hCD37-Mac5: (SEQ ID NO: 193)MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR and hCD37-Mac45:(SEQ ID NO: 194) MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNNLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARY.Furthermore, single point mutations are generated in the human largeextracellular loop sequence to identify residues important for antibodybinding.

Binding of CD37 binding agents to cells expressing SEQ ID NOs: 185-194is analyzed by flow cytometry as described above.

Example 14 Preparation of huCD37-3-SPP-DM1

The N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) linker wasdissolved in ethanol. The huCD37-3 antibody was incubated at 5 mg/mLwith a 7 fold molar excess of SPP linker for approximately 100 minutesat room temperature in 50 mM potassium phosphate buffer (pH 6.5)containing 50 mM NaCl, 2 mM EDTA, and 5% ethanol. The reaction mixturewas purified using a SEPHADEX™ G25F column equilibrated with theaforementioned potassium phosphate buffer. Antibody containing fractionswere pooled and used for subsequent steps.

The maytansinoid DM1 was dissolved in dimethylacetamide (DMA, finalconcentration is 3%) and a 1.7 fold molar excess relative to the linkerwas added drop wise to the SPP modified antibody. After overnightincubation at room temperature, the conjugated antibody was purified bychromatography on SEPHADEX™ G25F equilibrated in phosphate bufferedsaline (PBS), pH 6.5. The huCD37-3-SPP-DM1 conjugate was then dialyzedinto buffer containing 10 mM histidine, 250 mM glycine, 1% sucrose pH5.5. The number of DM1 molecules linked per antibody molecule wasdetermined using the previously reported extinction coefficients forantibody and DM1 (Liu et al., Proc. Natl. Acad. Sci. USA, 93, 8618-8623(1996)). The percentage of free maytansinoid present after theconjugation reaction was determined by injecting 20-50 μg conjugate ontoa HiSep column equilibrated in 25% acetonitrile in 100 mM ammoniumacetate buffer, pH 7.0, and eluting in acetonitrile. The peak area oftotal free maytansinoid species (eluted in the gradient and identifiedby comparison of elution time with known standards) was measured usingan absorbance detector set to a wavelength of 252 nm and compared withthe peak area related to bound maytansinoid (eluted in the conjugatepeak in the column flow-through fractions) to calculate the percentageof total free maytansinoid species. Conjugates with 3.5-4 DM1 moleculesper huCD37-3 antibody were obtained with <1% present as unconjugatedmaytansinoid.

Preparation of huCD37-3-SMCC-DM1

The (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC,Pierce Biotechnology, Inc) linker was dissolved in DMA. The huCD37-3antibody was modified with SMCC to introduce maleimides into theantibody by incubating the antibody at 5 mg/mL in 50 mM potassiumphosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5 with a 10 molar excess of SMCC.After approximately 100 minutes at ambient temperature, the reactionmixture was purified using a SEPHADEX™ G25 column equilibrated with thesame potassium phosphate buffer. Antibody containing fractions werepooled and used for subsequent steps.

The SMCC-modified antibody was reacted with a 10 mM solution of DM1 at a1.7 molar excess relative to the maleimide linker. The reaction wasstirred at ambient temperature under for approximately 18 hours. Theconjugation reaction mixture was filtered through a SEPHADEX™ G25 gelfiltration column equilibrated with 1×PBS at pH 6.5. ThehuCD37-3-SMCC-DM1 conjugate was then dialyzed into buffer containing 10mM histidine, 250 mM glycine, 1% sucrose pH 5.5. The number of DM1molecules linked per antibody molecule and the percentage of total freemaytansinoid species were determined as described above. Conjugates with3.5-4 DM1 molecules per huCD37-3 antibody were obtained with <1% presentas unconjugated maytansinoid.

Preparation of huCD37-3-Sulfo-Mal-DM4

Solutions of DM4 thiol and the heterobifunctional linker1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-(2,5-dioxopyrrolidin-1-yloxy)-4-oxobutane-2-sulfonicacid (3-sulfo-mal) were made up in N,N-dimethylacetamide (DMA) atconcentrations of 30-60 mM. The linker and DM4 were mixed together inDMA containing up to 40% v/v of 200 mM succinate buffer, 2 mM EDTA, pH5.0 to give a ratio of DM4 to linker of 1.6 and a final concentration ofDM4 equal to 15 mM. After mixing, the reaction was left for 2 h added toa mixture of huCD37-3 antibody in phosphate buffer (pH 7.5) under finalconjugation conditions of 4 mg/ml Ab, 90% phosphate buffer/10% DMA, pH7.5. The conjugation reaction was allowed to proceed at ambienttemperature for 2 h. The huCD37-3-sulfo-mal-DM4 conjugate was purifiedfrom excess unreacted DM4 and unconjugated linker products dialysis inPBS, followed by a final dialysis into buffer containing 10 mMhistidine, 250 mM glycine, 1% sucrose pH 5.5. The conjugate was filteredthrough a 0.22 μm filter for final storage. The number of DM4 moleculesper huCD37-3 antibody molecule (average) in the final conjugate thepercentage of total free maytansinoid species were determined asdescribed above. Conjugates with 3.5-4 DM4 molecules per huCD37-3antibody were obtained with <1% present as unconjugated maytansinoid.

Preparation of huCD37-3-PEG4-Mal-DM1

The N-hydroxysuccinimidyl-(polyethylene glycol)4-(N-maleimidomethyl)-DM1(NHS-PEG4-mal-DM1) reagent was dissolved in DMA. The huCD37-3 antibodywas incubated at 5 mg/mL in 50 mM potassium phosphate, 150 mM NaCl, 2 mMEDTA, pH 7.5 with a 7 fold molar excess of NHS-PEG4-mal-DM1 (10% DMAtotal). After approximately 2 hours at ambient temperature, the reactionmixture was purified using a SEPHADEX™ G25 column equilibrated in 1×PBS,pH 7.4. Antibody containing fractions were pooled and dialyzed intobuffer containing 10 mM histidine, 250 mM glycine, 1% sucrose, pH 5.5.The number of DM1 molecules linked per antibody and the percentage oftotal free maytansinoid species were determined as described above.Conjugates with 3.5-4 DM4 molecules per huCD37-3 antibody were obtainedwith <1% present as unconjugated maytansinoid.

Example 15 Binding Affinity of Maytansinoid Conjugates

Binding affinity of the exemplary huCD37-3 after conjugation toSMCC-DM1, SPP-DM1 or sulfo-mal-DM4 was assayed by flow cytometry asdescribed in the above example. The value for the apparent dissociationconstants (Kd) were calculated from the binding curves shown in FIG. 20Aand correspond to 0.26 nM for huCD37-3, 0.46 for huCD37-3-SMCC-DM1, 0.56nM for huCD37-3-SPP-DM1, and 0.89 nM for huCD37-3-sulfo-mal-DM4conjugates. This result demonstrates that SMCC-DM1, SPP-DM1 orsulfo-mal-DM4 conjugation does not notably alter the affinity of theexemplary huCD37-3 antibody.

Binding affinity of huCD37-38 after conjugation to SMCC-DM1 was assayedby flow cytometry as described in the above example. The value for theapparent dissociation constants (Kd) were calculated from the bindingcurves shown in FIG. 20B and correspond to 1.04 nM for huCD37-38 and 1.2nM for huCD37-38-SMCC-DM1 conjugates. This result demonstrates thatSMCC-DM1 conjugation does not notably alter the affinity of the huCD38antibody. Likewise, binding affinity of huCD37-50, huCD37-51, huCD37-56and huCD37-57 after conjugation to SMCC-DM1 was assayed by flowcytometry. The value for the apparent dissociation constants (Kd) werecalculated from binding curves and correspond to 0.43 nM for huCD37-50,0.70 nM for huCD37-50-SMCC-DM1, 2.0 nM for huCD37-51, 1.6 nM forhuCD37-51-SMCC-DM1, 0.3 nM for huCD37-56, 0.34 nM forhuCD37-56-SMCC-DM1, 0.30 for huCD37-57 and 0.34 for huCD37-57-SMCC-DM1.This result demonstrates that SMCC-DM1 conjugation also does not notablyalter the affinity of the huCD37-50, huCD37-51, huCD37-56 or huCD37-57antibodies.

Binding Affinity of PEG4-Mal-DM1 Conjugates

Binding affinity of the exemplary huCD37-3 and huCD37-50 antibodiesafter conjugation to PEG4-mal-DM1 was assayed by flow cytometry asdescribed in the above example. The value for the apparent dissociationconstants (Kd) were calculated from binding curves and correspond to0.28 nM for huCD37-3, 0.35 nM for huCD37-3-PEG4-mal-DM1, 0.68 nM forhuCD37-50 and 1.1 nM for huCD37-50-PEG4-mal-DM1 conjugates. This resultdemonstrates that PEG4-mal-DM1 conjugation does not notably alter theaffinity of the exemplary huCD37-3 or huCD50 antibodies.

Pro-Apoptotic Activity of huCD37-3-SMCC-DM1 Conjugates

Pro-apoptotic activity of huCD37-3 after conjugation to SMCC-DM1 wasevaluated by Annexin-V staining on Ramos cells as described above.Treatment with either huCD37-3 antibody or huCD37-3-SMCC-DM1 conjugateresulted in approximately 40% Annexin-V positive Ramos cells with anEC50 value of approximately 0.09 nM (FIG. 21A). Ramos cells treated witha non-binding control antibody or a non-binding SMCC-DM1 controlconjugate contained only up to 4% Annexin-V positive cells. Incomparison, treatment with the anti-CD20 antibody rituximab resulted inonly 16% Annexin-V positive cells with an EC50 value of approximately 2nM. In contrast, TRU-016 treatment did not increase the percentage ofAnnexin-V positive Ramos cells. This demonstrates that the strongpro-apoptotic activity of the human anti-CD37 antibody huCD37-3 isretained after conjugation to SMCC-DM1.

CDC Activity of huCD37-3-SMCC-DM1 Conjugates

CDC activity of huCD37-3 after conjugation to SMCC-DM1 was evaluated onRamos cells in the presence of 5% human complement. Treatment withhuCD37-3 or huCD37-3-SMCC-DM1 resulted in a reduction in cell viabilityto 53% and 73%, respectively (FIG. 21B). Therefore, the CDC activity ofthe exemplary huCD37-3 antibody is maintained after maytansinoidconjugation.

ADCC Activity of Conjugates

ADCC activity of huCD37-3 after conjugation to SMCC-DM1 or PEG4-mal-DM1was evaluated on Daudi and Ramos cells in the presence of human NKeffector cells by LDH release assay as described above. As can be seenin FIG. 22A, huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates havesimilar ADCC activity as the unconjugated huCD37-3 antibody on Daudicells. Treatment with huCD37-3, huCD37-3 SMCC-DM1 orhuCD37-3-PEG4-mal-DM1 resulted in approximately 41%, 39% or 36% Daudicell lysis with an EC50 value of 0.42 ng/mL, 1.13 ng/mL, or 0.91 ng/mL,respectively. Similar results were obtained using Ramos cells as targetcells. As before, huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugateshave comparable ADCC activity to the unconjugated huCD37-3 antibody onRamos cells (FIG. 22B). Treatment with huCD37-3, huCD37-3 SMCC-DM1 orhuCD37-3-PEG4-mal-DM1 resulted in approximately 43%, 41% or 42% Ramoscell lysis with an EC50 value of 0.95 ng/mL, 1.33 ng/mL, or 1.57 ng/mL,respectively. Therefore, the potent ADCC activity of the exemplaryhuCD37-3 antibody is maintained after maytansinoid conjugation.

Induction of Cell Cycle Arrest by huCD37-3-SMCC-DM1

The potential of anti-CD37 antibodies and conjugates to induce cellcycle arrest in cell lines was evaluated by propridium iodide (PI)staining followed by flow cytometry analysis. Exponentially growingcells were harvested by centrifugation at 1,300 rpm for 5 minutes at RTand resuspended at 0.5×106 cells/mL in complete RPMI media. Cells weretransferred at 1 mL per well to a 24-well plate (Falcon 3077) to equal0.5×10⁶ cells/assay. The test compounds were added to each well in afinal concentration of 10 nM. Complete RPMI media was added to untreatedcontrol wells. Cells were incubated overnight for 16 to 20 hrs at 37° C.in a humidified 5% CO₂ incubator. The next day, cells were harvested bytransferring into 5 mL polystyrene tubes, washed once with 3 mL PBS, andfixed in 1 mL 70% ethanol for 30 minutes on ice. The samples were thenwashed again with 3 mL PBS once and resuspended in 0.5 mL PBS. RNase wasadded to the sample at 5 μL/mL and incubated at 37° C. for 30 minutes.The samples were then stained with propidium iodide at a finalconcentration of 50 μg/mL. Samples were acquired within 24 hours of PIstaining. Samples were run on a FACS Calibur (BD Biosciences, SanDiego). The FL2-A parameter was set to linear scale and the FL2 PTM wasadjusted to position the G1 peak around 200. Samples were acquired at alow flow rate and 10,000 events were collected per sample. Distributionof cells in the different phases of the cell cycle was determined usingModFit software (Version 5.11, Verity Software House Inc., USA). Thisprogram utilizes peak fitting techniques to automatically model the PIdata and provides the desired quantitative data. The data was analyzedwith standard program settings.

The effect of huCD37-3 and huCD37-3-SMCC-DM1 on cell cycle arrest ofBJAB and RL lymphoma cells was evaluated after a 16-20 hour incubationwith either compound at a 10 nM concentration followed by propridiumiodide (PI) staining and flow cytometry analysis. Incubation withhuCD37-3-SMCC-DM1 resulted in an increase in the percentage of cells inG2/M phase from 13% for untreated BJAB lymphoma cells to 95% forhuCD37-3-SMCC-DM1 treated cells (FIG. 23A). Similarly, incubation withhuCD37-3-SMCC-DM1 resulted in an increase in the percentage of cells inG2/M phase from 12% for untreated RL lymphoma cells to 33% forhuCD37-3-SMCC-DM1 treated cells (FIG. 23B). In contrast, the huCD37-3antibody had no effect on the cell cycles of either BJAB or RL cells. Inaddition, a non-binding SMCC-DM1 conjugate tested at the sameconcentration also had no effect on the cell cycle of either cell type.This demonstrated that maytansinoid conjugates made with isolatedanti-CD37 antibodies caused specific cell cycle arrest of CD37-positivelymphoma cell lines.

Example 16 In Vitro Cytotoxicity Assays

The ability of antiCD37 antibody-conjugates to inhibit cell growth wasmeasured using in vitro cytotoxicity assays as described in Example 10for antibodies. Briefly, target cells were plated at 5,000 cells perwell in 100 μL in complete RPMI media (RPMI-1640, 10% fetal bovineserum, 2 mM glutamine, 1% penicillin-streptomycin, all reagents fromInvitrogen). Conjugates were diluted into complete RPMI media using3-fold dilution series and 100 μL were added per well. The finalconcentration typically ranged from 3×10⁻⁸ M to 4.6×10⁻¹² M. Cells wereincubated at 37° C. in a humidified 5% CO₂ incubator for 4 to 5 days.Viability of remaining cells was determined by colorimetric WST-8 assayas described for antibody assays and the absorbance at 450 nm (A450) wasmeasured in a multiwell plate reader (Dojindo Molecular Technologies,Inc., Rockville, Md., US). The percent viability was calculated bydividing each treated sample value by the average value of wells withuntreated cells. The percent viability value was plotted against theantibody-conjugate concentration in a semi-log plot for each treatment.

In Vitro Cytotoxicity of SMCC-DM1 Conjugates of Various Antibodies

The in vitro cytotoxicity of SMCC-DM1 conjugates made with variousanti-CD37 antibodies was compared to the activity of a non-specifichuIgG-SMCC-DM1 conjugate. The results from a typical cytotoxicity assayare shown in FIG. 24A for Daudi cells incubated with huCD37-3-SMCC-DM1,huCD37-38-SMCC-DM1, huCD37-50-SMCC-DM1, huCD37-51-SMCC-DM1,huCD37-56-SMCC-DM1, huCD37-57-SMCC-DM1, or a non-binding huIgG1-SMCC-DM1control conjugate. All specific conjugates resulted in specific cellkilling as compared to the control conjugate and reduced the cellviability completely at the highest concentration tested. The EC50values correspond to 0.067 nM, 0.098 nM, 0.13 nM, 0.20 nM, 0.31 nM and0.35 nM for SMCC-DM1 conjugates of huCD37-3, huCD37-38, huCD37-50,huCD37-51, huCD37-56 and huCD37-57, respectively. In contrast, SMCC-DM1conjugates of a non-binding isotype control antibody resulted in cellkilling with an EC50 value of only 20 nM.

Likewise, FIG. 24B shows the results of a typical cytotoxicity assayusing Granta-519 cells incubated with huCD37-3-SMCC-DM1,huCD37-38-SMCC-DM1, huCD37-50-SMCC-DM1, huCD37-51-SMCC-DM1, or anon-binding huIgG1-SMCC-DM1 control conjugate for 5 days. Treatment withall specific SMCC-DM1 completely reduced viability at the highestconcentration tested with an EC50 of 0.047 nM, 0.074 nM, 0.12 nM and0.25 nM for SMCC-DM1 conjugates of huCD37-3, huCD37-38, huCD37-50, andhuCD37-51, respectively. In contrast, SMCC-DM1 conjugates of anon-binding isotype control antibody resulted in cell killing with anEC50 value of only 20 nM.

In Vitro Cytotoxicity of huCD37-3-SMCC-DM1, —SPP-DM1 and Sulfo-Mal-DM4Conjugates

The in vitro cytotoxicity of huCD37-3-SMCC-DM1, —SPP-DM1 andsulfo-mal-DM4 conjugates against Daudi, Granta-519 and BJAB cells wascompared to the activity of a non-specific huIgG-MCC-DM1 conjugate. Allconjugates tested reduced viability of Daudi cells completely at thehighest concentration tested with an EC50 value of 0.065 nM, 0.12 nM and0.14 nM for huCD37-3-SMCC-DM1, huCD37-3-SPP-DM1 andhuCD37-3-sulfo-mal-DM4, respectively. In contrast, the non-specifichuIgG-SMCC-DM1 conjugate had an EC50 of 19 nM. Likewise, all conjugatestested reduced viability of Granta-519 cells completely at the highestconcentration tested with an EC50 value of 0.047 nM, 0.13 nM and 0.088nM for huCD37-3-SMCC-DM1, huCD37-3-SPP-DM1 and huCD37-3-sulfo-mal-DM4,respectively. In contrast, the non-specific huIgG-SMCC-DM1 conjugate hadan EC50 of 19 nM. Finally, all conjugates tested reduced viability ofBJAB cells completely at the highest concentration tested with an EC50value of 0.041 nM, 0.11 nM and 0.11 nM for huCD37-3-SMCC-DM1,huCD37-3-SPP-DM1 and huCD37-3-sulfo-mal-DM4, respectively. In contrast,the non-specific huIgG-SMCC-DM1 conjugate had an EC50 of 16 nM.

In Vitro Cytotoxicity of huCD37-3-SMCC-DM1 and huCD37-3-PEG4-Mal-DM1Conjugates

The in vitro cytotoxicity of huCD37-3-SMCC-DM1 and huCD37-3-PEG4-mal-DM1conjugates against a panel of lymphoma cell lines was compared to theactivity of a non-specific huIgG-SMCC-DM1 conjugate. Treatment withhuCD37-3 conjugates completely reduced Daudi cell viability at thehighest concentration tested with an EC50 of 0.036 nM or 0.018 nM forhuCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates, respectively. Incontrast, the non-binding isotype control conjugates reduced viabilitywith an EC50 of 16 nM or greater than 30 nM for huIgG-SMCC-DM1 orhuIgG-PEG4-mal-DM1, respectively. Likewise, treatment with huCD37-3conjugates completely reduced Granta-519 cell viability at the highestconcentration tested with an EC50 of 0.014 nM or 0.012 nM forhuCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates, respectively. Incontrast, the non-binding isotype control conjugates reduced viabilitywith an EC50 of 6.5 nM or greater than 12 nM for huIgG-SMCC-DM1 orhuIgG-PEG4-mal-DM1, respectively.

The in vitro cytotoxicity of huCD37-3-SMCC-DM1 and huCD37-3-PEG4-mal-DM1conjugates against a panel of lymphoma cell lines was compared to theactivity of a non-specific huIgG-SMCC-DM1 conjugate. Treatment withhuCD37-3 conjugates completely reduced BJAB cell viability at thehighest concentration tested with an EC50 of 0.019 nM or 0.010 nM forhuCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates, respectively. Incontrast, the non-binding isotype control conjugates reduced viabilitywith an EC50 of 13 nM or 17 nM for huIgG-SMCC-DM1 or huIgG-PEG4-mal-DM1,respectively. Likewise, treatment with huCD37-3 conjugates completelyreduced SU-DHL-4 cell viability at the highest concentration tested withan EC50 of 0.031 nM or 0.024 nM for huCD37-3-SMCC-DM1 orhuCD37-3-PEG4-mal-DM1 conjugates, respectively. In contrast, thenon-binding isotype control conjugates reduced viability with an EC50 ofgreater than 30 nM for both huIgG-SMCC-DM1 or huIgG-PEG4-mal-DM1. ThehuCD37-3-SMCC-DM1 conjugate also showed potency against the FL cell lineDOHH-2 as well as CLL cell lines such as JVM-2 and JVM-3 (FIG. 32B).

Treatment with huCD37-3 conjugates completely reduced Raji cellviability at the highest concentration tested with an EC50 of 0.071 nMor 0.045 nM for huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates,respectively. In contrast, the non-binding isotype control conjugatesreduced viability with an EC50 of 24 nM or 47 nM for huIgG-SMCC-DM1 orhuIgG-PEG4-mal-DM1, respectively. Next, the same conjugates were testedin a vincristine-resistant Raji clone termed Raji-VCR. As seen for theparental Raji cell, both conjugates showed specific cell killingTreatment with huCD37-3 conjugates completely reduced Raji-VCR cellviability at the highest concentration tested with an EC50 of 0.11 nM or0.037 nM for huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates,respectively. In contrast, the non-binding isotype control conjugatesreduced viability with an EC50 of 46 nM or 100 nM for huIgG-SMCC-DM1 orhuIgG-PEG4-mal-DM1, respectively.

Treatment with huCD37-3 conjugates completely reduced Namalwa cellviability at the highest concentration tested with an EC50 of 0.033 nMor 0.024 nM for huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates,respectively. In contrast, the non-binding isotype control conjugatesreduced viability with an EC50 of 20 nM or greater than 30 nM forhuIgG-SMCC-DM1 or huIgG-PEG4-mal-DM1, respectively. Likewise, treatmentwith huCD37-3 conjugates completely reduced Ramos cell viability at thehighest concentration tested with an EC50 of 0.16 nM or 0.069 nM forhuCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1 conjugates, respectively. Incontrast, the non-binding isotype control conjugates reduced viabilitywith an EC50 of 20 nM for huIgG-SMCC-DM1 and greater than 30 nM forhuIgG-PEG4-mal-DM1.

In Vitro Cytotoxicity of huCD37-3-SMCC-DM1 on Antigen Negative Molt-4Cells

To further verify the specificity of huCD37-3-SMCC-DM1 cytotoxicity, itsactivity was compared to a non-specific huIgG-MCC-DM1 conjugate againstnon-CD37 expressing Molt-4 T-cell acute lymphoblastic leukemia cellline. An increased concentration of both conjugates was used in thisexperiment to capture the relatively poor non-specific cytotoxicity.HuCD37-3-SMCC-DM1 and the non-specific conjugate showed the samecytotoxicity with an EC50 of 38 nM and 42 nM, respectively.

Summary of In Vitro Cytotoxicity of Anti-CD37 Antibody MaytansinoidConjugates

Taken these cytotoxicity results together, it is apparent thatconjugates made with the isolated anti-CD37 antibodies showed specificcytotoxicity against a panel of CD37-positive lymphoma cell lines (FIG.32B). In each case tested a good specificity window is observed for eachCD37-expressing cell line, suggesting that cytotoxicity is a result ofspecific anti-CD37 antibody binding to target cells. In addition,huCD37-3-SMCC-DM1 and the non-specific conjugate showed the same poorcytotoxicity against antigen-negative Molt-4 cells. This demonstratesthat the cytotoxicity observed for this exemplary conjugate is dependenton CD37 expression. The huCD37-3 antibody was also active against manycell lines including DOHH-2, Granata-519, SU-DHL-4, JVM-2, and JVM-3. Incontrast, the anti-CD37 SMIP TRU-016 compound had no direct effect onsurvival of any of these cell lines. The anti-CD20 antibody showed lessdirect activity than huCD37-3 in most of these cell lines despite theoften higher CD20 expression levels as measured by quantitative flowcytometry (FIG. 32A).

Example 17 In Vivo Efficacy of Anti-CD37 Antibodies and their SMCC-DM1Conjugates in a BJAB Xenograft Model

Anti-CD37 antibodies and their SMCC-DM1 conjugates were tested in anestablished xenograft model using BJAB lymphoma cells implantedsubcutaneous into SCID mice. Animals were randomized by tumor volumeinto treatment groups when tumors reached a mean tumor volume ofapproximately 120 mm³ and treated once on day 12 post cell inoculationwith either 10 mg/kg of (A) huCD37-3 Ab, huCD37-3-SMCC-DM1, huCD37-50Ab, huCD37-50-SMCC-DM1 or (B) huCD37-38 Ab, huCD37-38-SMCC-DM1,huCD37-56 Ab, huCD37-56-SMCC-DM1. The mean tumor volume of the differenttreatment groups is plotted against time post tumor cell inoculation inFIG. 25. It is apparent that treatment with any of the antibodiesresulted in a moderate reduction in mean tumor volume, while treatmentwith any of the SMCC-DM1 conjugates resulted in a more significantreduction in mean tumor volume. In addition, for each treatment a % T/Cvalue was calculated which corresponds to the median tumor volume ofeach treated group divided by the median tumor volume of the vehicletreated group. A treatment with a % T/C value of below 42% is consideredactive, while a treatment with a % T/C value of below 12% is consideredhighly active. Treatment with all SMCC-DM1 conjugates tested resulted ina significant reduction in median tumor volume. The % T/C value on day29 post cell inoculation corresponded to 20%, 20%, 9% or 4% forhuCD37-3-SMCC-DM1, huCD37-50-SMCC-DM1, huCD37-38-SMCC-DM1 orhuCD37-56-SMCC-DM1, respectively.

In Vivo Efficacy of huCD37-3 Antibody, Sulfo-Mal-DM4, —SPP-DM1 andSMCC-DM1 Conjugates in a BJAB Xenograft Model

In order to evaluate the in vivo efficacy of additional maytansioidconjugates, the sulfo-mal-DM4 and SPP-DM1 conjugates of the exemplaryhuCD37-3 antibody were compared to SMCC-DM1 conjugates in a xenograftmodel using BJAB lymphoma cells implanted intravenously into SCID mice.

Animals were randomized by tumor volume into treatment groups whentumors reached a mean tumor volume of approximately 120 mm³ and treatedonce on day 9 post cell inoculation with either 10 mg/kg of huCD37-3 Ab,huCD37-3-SMCC-DM1, huCD37-3-sulfo-mal-DM4 or 5 mg/kg ofhuCD37-3-SPP-DM1. The mean tumor volume of the different treatmentgroups is plotted against time post tumor cell inoculation in FIG. 26.It is apparent that treatment with any of the conjugates resulted in asignificant reduction in mean tumor volume. The % T/C value wascalculated as described above for each treatment using the median tumorvolume for each treatment group. The % T/C value on day 21 post cellinoculation corresponded to 49%, 5%, 7% or 4% for huCD37-3,huCD37-3-SMCC-DM1, huCD37-3-sulfo-mal-DM4 or huCD37-3-SPP-DM1,respectively. At the end of the study on day 121, huCD37-3-sulfo-mal-DM4treatment resulted in 3 of 8 tumor-free survivors (TFS), whilehuCD37-3-SPP-DM1 treatment resulted in 1 of 8 TFS. No TFS were observedin the huCD37-3 antibody, huCD37-3-SMCC-DM1 or PBS vehicle controlgroups. This indicated that maytansinoid conjugates of the huCD37-3antibody, such as for example SMCC-DM1, sulfo-mal-DM4 or SPP-DM1conjugates, were highly active in the BJAB model.

In Vivo Efficacy of huCD37-3 Antibody, Sulfo-Mal-DM4, —SPP-DM1 andSMCC-DM1 Conjugates in a SU-DHL-4 Xenograft Model

A second xenograft model using SU-DHL-4 diffuse large B-cell lymphomacells implanted subcutaneous into SCID mice was utilized to evaluate thein vivo efficacy of the sulfo-mal-DM4 and SPP-DM1 conjugates of theexemplary huCD37-3 antibody as compared to SMCC-DM1 conjugates Animalswere randomized by body weight into treatment groups when tumors wereestablished and treated once on day 17 post cell inoculation with either10 mg/kg of huCD37-3 Ab, huCD37-3-SMCC-DM1, huCD37-3-sulfo-mal-DM4 or 5mg/kg of huCD37-3-SPP-DM1. The median tumor volume of the differenttreatment groups is plotted against time post tumor cell inoculation inFIG. 27. It is apparent that treatment with the huCD37-3 antibodyresulted in a reduction in median tumor volume, while treatment with anyof the conjugates resulted in a more significant reduction in mediantumor volume. The % T/C value was calculated as described above for eachtreatment. The % T/C value on day 37 post cell inoculation correspondedto 32%, 1%, 1% or 3% for huCD37-3, huCD37-3-SMCC-DM1,huCD37-3-sulfo-mal-DM4 or huCD37-3-SPP-DM1, respectively. At the end ofthe study on day 125, huCD37-3-SMCC-DM1 or huCD37-3-sulfo-mal-DM4treatment resulted in 8 of 10 tumor-free survivors (TFS), whilehuCD37-3-SPP-DM1 treatment resulted in 9 of 10 TFS. No TFS were observedin the huCD37-3 antibody or PBS vehicle control groups. This indicatedthat the huCD37-3 antibody itself was active with a single 10 mg/kg dosein the SU-DHL-4 model. In addition, maytansinoid conjugates, such as forexample SMCC-DM1, sulfo-mal-DM4 or SPP-DM1 conjugates, added efficacy tothe antibody and result in even greater potency in this model.

In Vivo Efficacy of huCD37-3 Antibody, PEG4-Mal-DM1 and SMCC-DM1Conjugates in a BJAB Xenograft Model

The huCD37-3 antibody and its PEG4-mal-DM1 and SMCC-DM1 conjugates weretested in an established xenograft model using BJAB lymphoma cellsimplanted subcutaneous into SCID mice. Animals were randomized by tumorvolume into treatment groups when tumors reached a mean tumor volume ofapproximately 120 mm³ and treated once on day 9 post cell inoculationwith either 10 mg/kg of huCD37-3 Ab, huCD37-3-SMCC-DM1 orhuCD37-3-PEG4-mal-DM1. As seen in FIG. 28, treatment with eitherconjugate resulted in a significant reduction in mean tumor volume. The% T/C value was calculated as described above for each treatment usingthe median tumor volume for each treatment group. The % T/C value on day24 post cell inoculation corresponded to 48%, 16% or 5% for huCD37-3,huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1, respectively. On day 74 postcell inoculation, huCD37-3-SMCC-DM1 treatment resulted in 1 of 9tumor-free survivors (TFS), while huCD37-PEG4-mal-DM1 treatment resultedin 1 of 9 TFS. No TFS were observed in the huCD37-3 antibody or PBSvehicle control groups. In addition, huCD37-3-SMCC-DM1 was also activeat a single dose of 5 mg/kg in this model with a % T/C value on day 24post cell inoculation of 34%. This indicated that maytansinoidconjugates of the huCD37-3 antibody, such as for example SMCC-DM1 orPEG4-mal-DM1 conjugates, were highly active in the BJAB model.

In Vivo Efficacy of huCD37-3 Antibody, PEG4-Mal-DM1 and SMCC-DM1Conjugates in a SU-DHL-4 Xenograft Model

The huCD37-3 antibody and its PEG4-mal-DM1 and SMCC-DM1 conjugates weretested in an established xenograft model using SU-DHL-4 diffuse largeB-cell lymphoma cells implanted subcutaneous into SCID mice. Animalswere randomized by body weight into treatment groups and treated once onday 15 post cell inoculation with either 10 mg/kg of huCD37-3 Ab,huCD37-3-SMCC-DM1 or huCD37-3-PEG4-mal-DM1. The mean tumor volume of thedifferent treatment groups is plotted against time post tumor cellinoculation FIG. 29. It is apparent that treatment with the huCD37-3antibody resulted in a reduction in mean tumor volume, while treatmentwith either conjugate resulted in a more significant reduction in meantumor volume. The % T/C value was calculated as described above for eachtreatment using the median tumor volume for each treatment group. The %T/C value on day 38 post cell inoculation corresponded to 34%, 4% or 2%for huCD37-3, huCD37-3-SMCC-DM1, or huCD37-3-PEG4-mal-DM1, respectively.On day 74 post cell inoculation, huCD37-3-SMCC-DM1 treatment resulted in8 of 10 tumor-free survivors (TFS), while huCD37-3-PEG4-mal-DM1treatment resulted in 10 of 10 TFS. No TFS were observed in the huCD37-3antibody or PBS vehicle control groups. This indicated that the huCD37-3antibody itself was active with a single 10 mg/kg dose in the SU-DHL-4model. In addition, maytansinoid conjugates, such as for exampleSMCC-DM1 or PEG4-mal-DM1 conjugates, showed enhanced efficacy ascompared to the unconjugated antibody efficacy to the antibody andresulted in even greater potency in this model. The huCD37-3-SMCC-DM1conjugate also showed strong efficacy at single doses of 2.5 or 5 mg/kgin this model with % T/C values on day 37 post cell inoculation of 18%and 6%, respectively.

In Vivo Efficacy of huCD37-3 Antibody, huCD37-3-SMCC-DM1 Conjugate,Rituximab Antibody, and a Regime of Cyclophosphamide, Vincristine, andPrednisone (CVP) in a DoHH2 Xenograft Model

The huCD37-3 antibody and its SMCC-DM1 conjugate were tested in anestablished xenograft model using DoHH2 follicular lymphoma cellsimplanted subcutaneously into SCID mice. Animals were randomized bytumor volume into treatment groups, and treatments started on day 12post inoculation with either a single dose of 10 mg/kg of huCD37-3antibody or huCD37-3-SMCC-DM1 conjugate; six doses of 2 mg/kg ofRituximab twice per week for three weeks; or with a regimen of a single40 mg/kg dose of cyclophosphamide, and 0.5 mg/kg of vincristine, alongwith five daily 0.2 mg/kg doses of prednisone (CVP). The median tumorvolume of the different treatment groups was plotted against time posttumor cell inoculation in FIG. 30. Treatment with the huCD37-3 antibodyresulted in a reduction in median tumor volume, while treatment withhuCD37-3-SMCC-DM1 conjugate resulted in a more significant reduction inmedian tumor volume. The huCD37-3-SMCC-DM1 conjugate resulted in amedian tumor reduction similar to treatment with Rituximab and a moredurable median tumor reduction as compared to treatment with CVP. Thetumor growth delay (T-C value) was defined as the median time (in days),required for the treatment group (T) and the control group (C) tumors toreach a predetermined size and was calculated for each treatment groupexcluding the tumor free survivors. The T-C value for median treatmenttumors to reach 800 mm³ corresponded to 8, 25, 24, and 13 days forhuCD37-3, huCD37-3-SMCC-DM1, rituximab and CVP, respectively. At the endof the study, on day 130 post cell inoculation, huCD37-3-SMCC-DM1treatment resulted in 1 of 9 tumor-free survivors (TFS). No TFS wereobserved in the huCD37-3 antibody, rituximab, CVP, or PBS vehiclecontrol groups. The SMCC-DM1 conjugate showed comparable tumor growthdelay to rituximab and enhanced tumor growth delay as compared to theunconjugated antibody and treatment with CVP in the DoHH2 model.

In Vivo Efficacy of huCD37-3 Antibody, huCD37-3-SMCC-DM1 Conjugate,Ofatumumab Antibody and Bendamustine in a JVM-3 Xenograft Model

The huCD37-3 antibody and its huCD37-3-SMCC-DM1 conjugate were tested inan established xenograft model using JVM-3 chronic lymphocytic leukemiacells implanted subcutaneously into SCID mice. Animals were randomizedby tumor volume into treatment groups, and treatments started on day 7post inoculation with either a single dose of 10 mg/kg of huCD37-3antibody, a 5 or a 10 mg/kg dose of huCD37-3-SMCC-DM1 conjugate, sixdoses of 5 mg/kg of ofatumumab twice per week for three weeks, or asingle 50 mg/kg dose of bendamustine. The median tumor volume of thedifferent treatment groups was plotted against time post tumor cellinoculation in FIG. 31. Treatment with the huCD37-3 antibody resulted ina reduction in median tumor volume, while treatment withhuCD37-3-SMCC-DM1 conjugate resulted in a more significant reduction inmedian tumor volume. The % T/C value was calculated as described abovefor each treatment using the median tumor volume for each treatmentgroup. The % T/C value on day 20 post cell inoculation corresponded to31%, 19%, 10%, 35%, and 38% for huCD37-3, 5 mg/kg huCD37-3-SMCC-DM1, 10mg/kg huCD37-3-SMCC-DM1, ofatumumab, and bendamustine, respectively. Atthe end of the study, on day 76 post cell inoculation, huCD37 andofatumumab antibody treatments both resulted in 1 of 10 tumor-freesurvivors (TFS), while huCD37-3-SMCC-DM1 at 5 and 10 mg/kg resulted in 1and 2 out of 10 TFS, respectively. No TFS were observed in thebendamustine or PBS vehicle control groups. This indicated that thehuCD37-3 antibody itself was active at a single 10 mg/kg dose in theJVM-3 model. The maytansinoid conjugate, huCD37-3-SMCC-DM1, showedenhanced efficacy as compared to the unconjugated antibody. In addition,treatment with the huCD37-3-SMCC-DM1 maytansinoid conjugate resulted ineven greater potency than ofatumumab or bendamustine treatment in thismodel.

Summary of In Vivo Efficacy of Anti-CD37 Antibody Conjugates

CD37 has not been evaluated as a target for maytansinoidimmunoconjugates, however CD20 has. CD37 is structurally similar to CD20as both antigens are cell surface proteins that contain 4 transmembranedomains and one small and one large extracellular loop. Antibodiesagainst either antigen have been shown to be internalized slowly andhave a slow to moderate rate of intracellular metabolism (Press et al.1989, Cancer Res. 49(17):4906-12, and Press et al. 1994, Blood.83(5):1390-7). Immunoconjugates of CD20 antibodies have been evaluatedpreviously. In one case, non-cleavable MCC-DM1 conjugates of ananti-CD20 antibody showed the same efficacy as the unconjugatedantibody, while a cleavable SPP-DM1 conjugate of the same antibodyshowed improved efficacy in a Granta-519 xenograft model in SCID mice(Polson A G, Cancer Res 2009; 69:2358-64). Similarly, calicheamicinconjugates of rituximab made with an acid-stable amide linker were didnot show improved in vivo efficacy in a Ramos xenograft model in nudemice. Only calicheamicin conjugates of rituximab made with anacid-labile dimethyl hydrazide Ac-But linker showed improved in vivoefficacy in this study (DiJoseph J F, Cancer Immunol Immunotherapy 2007;56:1107-1117).

In striking contrast non-cleavable SMCC-DM1 conjugates of severalisolated anti-CD37 antibodies of this invention show dramaticallyimproved in vivo efficacy in BJAB, SU-DHL-4, DoHH2 and JVM-3 xenograftmodel as compared to the unconjugated antibody. This suggests that theisolated antibodies have unique properties that allow them to be moreefficacious as maytansinoid conjugates, such as for example SMCC-DM1conjugates, in vivo.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections sets forth one or more,but not all, exemplary embodiments of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

What is claimed is:
 1. An antibody produced by the hybridoma of ATCC Deposit Designation PTA-10664 or an antigen-binding fragment thereof.
 2. An isolated antibody or antigen binding fragment thereof that specifically binds to CD37, wherein said antibody, or antigen binding fragment comprises the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 polypeptide sequences of SEQ ID NOs: 4, 5, and 6 and SEQ ID NOs: 28, 29, and 30 respectively.
 3. An isolated cell producing the antibody or antigen binding fragment thereof of claim
 2. 4. A method of making an anti-CD37 antibody or antigen-binding fragment thereof comprising (a) culturing the cell of claim 3; and (b) isolating said antibody, or antigen-binding fragment thereof from said cultured cell.
 5. An immunoconjugate having the formula (A)-(L)-(C), wherein: (A) is the antibody or antigen binding fragment of claim 2; (L) is a linker; and (C) is a cytotoxic agent; and wherein said linker (L) links (A) to (C).
 6. The immunoconjugate of claim 5 comprising 3-4 (C) per (A).
 7. The immunoconjugate of claim 5, wherein said linker is selected from the group consisting: N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfoSMCC); N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); and N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester (NHS-PEG4-maleimide).
 8. The immunoconjugate of claim 5, wherein said cytotoxic agent is selected from the group consisting of a maytansinoid, maytansinoid analog, doxorubicin, a modified doxorubicin, benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin, duocarmycin analog, calicheamicin, dolastatin, dolastatin analog, aristatin, tomaymycin derivative, and leptomycin derivative or a prodrug of the agent.
 9. The immunoconjugate of claim 8, wherein said cytotoxic agent is N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) or N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).
 10. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of claim 2 and a pharmaceutically acceptable carrier.
 11. A pharmaceutical composition comprising the immunoconjugates of claim 5, wherein the immunoconjugates have an average of about 3 to about 4 (C) per (A).
 12. A diagnostic reagent comprising the antibody or antigen binding fragment thereof of claim
 2. 13. A kit comprising the antibody or antigen binding fragment thereof of claim
 2. 14. The immunoconjugate of claim 5, wherein the antibody comprises the polypeptide sequences of SEQ ID NO:57 and SEQ ID NO:74.
 15. The antibody or antigen binding fragment of claim 2, wherein the antibody or antigen binding fragment comprises the polypeptide sequences of SEQ ID NO:57 and SEQ ID NO:74.
 16. The immunoconjugate of claim 5, wherein said linker is SMCC.
 17. The immunoconjugate of claim 16, wherein said cytotoxic agent is N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1).
 18. The immunoconjugate of claim 14, wherein said linker is SMCC.
 19. The immunoconjugate of claim 14, wherein said cytotoxic agent is N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1).
 20. The immunoconjugate of claim 18, wherein said cytotoxic agent is N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1).
 21. An immunoconjugate having the formula (A)-(L)-(C), wherein: (A) is the antibody or antigen binding fragment of claim 15; (L) is a linker; and (C) is a cytotoxic agent; and wherein said linker (L) links (A) to (C).
 22. A pharmaceutical composition comprising the antibody or antigen binding fragment of claim 15 and a pharmaceutically acceptable carrier.
 23. A pharmaceutical composition comprising the immunoconjugate of claim 17 and a pharmaceutically acceptable carrier.
 24. A pharmaceutical composition comprising the immunoconjugate of claim 20 and a pharmaceutically acceptable carrier.
 25. A pharmaceutical composition comprising the immunoconjugates of claim 17, wherein the immunoconjugates have an average of about 3 to about 4 (C) per (A).
 26. A pharmaceutical composition comprising the immunoconjugates of claim 20, wherein the immunoconjugates have an average of about 3 to about 4 (C) per (A). 